Ia Determinants Research Articles

Genetic control of the immune response to haemoglobin. III. Variant A beta (bm12) but not Ae (D2.GD) Ia polypeptides alter immune responsiveness towards the alpha -subunit of human haemoglobin.

Mice bearing the I-A subregion mutation bm12 were immunized and challenged with the alpha -subunit of human adult haemoglobin. Under conditions in which parental B6/Kh mice respond, B6.C-H-2bm12 mice are inhibited nearly 100% in their ability to respond to challenge to the alpha-chain of haemoglobin. D2.GD mice which express a variant Ae (E beta) polypeptide of the I-E subregion can respond as well as B10.D2 mice to both subunits (alpha- and beta-) of haemoglobin. These observations as well as other genetic mapping data confirm the I-A mapping of alpha -chain-specific Ir genes and extend the genetic fine mapping to the A beta gene within the I-A subregion or a combinatorial Ia determinant generated by an interaction of A alpha and A beta. In addition they implicate the Ia.8 specificity in determining immune responsiveness to alpha -chain determinants.

Active and late rosette-forming cells: Immunological and cytochemical characterization

In order to characterize T-cell subpopulations with different affinities for sheep red blood cells, a possible correlation between active and late rosette-forming cells (RFC), expression of Fc receptors, and the presence of Ia antigens was investigated. Further analysis of their spontaneous in vitro DNA synthesis, and their cytochemical pattern was also performed. Most of the active T cells lacked Fc receptors and expressed Ia antigens, whereas the large majority of cells with Fc-IgG and -IgM receptors were included as late T cells, but lacked Ia determinants. Analysis of spontaneous blastogenesis showed that late RFC, but not active RFC, synthesize DNA in unstimulated cultures. Cytochemical analysis suggested that Active RFC display a more immature phenotype compared to late RFC, which have enzymatic markers present in mature T cells. Possible interpretations of these findings are discussed.

Characterization of resident glomerular cells in the rat expressing Ia determinants and manifesting genetically restricted interactions with lymphocytes.

The existence of a subpopulation of rat glomerular cells bearing Ia determinants has been demonstrated with the aid of techniques for the enzymatic isolation and culture of glomerular cells. The Ia-positive cell is normally resident in the uninflamed glomerulus. It resembles a mononuclear phagocyte and consists of a functionally heterogeneous cell population with the capacity of Fc receptor display and phagocytosis, both in vivo and in vitro. A new technique for labeling these cells in situ in intact glomeruli has indicated that Ia-positive cells make up approximately 2% of the total glomerular cell population. The isolated glomerular cells can take up antigen and stimulate immune lymphocytes in an I-region-restricted interaction. They are strongly stimulatory in an allogeneic primary mixed lymphocyte culture. Characterization of this cell type suggests potential new insights into the pathogenesis of renal allograft rejection and immunologically mediated glomerulonephritis.

Role of accessory cells in B cell activation. IV. Ia+ accessory cells are required for the in vitro generation of thymic independent type 2 antibody responses to polysaccharide antigens.

It has been previously shown that the in vitro antibody response to TNP-Ficoll requires the presence of adherent accessory cells. In order to determine if this characteristic was unique to TNP-Ficoll or a general feature of the TI-2 antibody responses, responses to the polysaccharide antigens TNP-Levan and TNP-Dextran were studied. Also, it was determined if the functionally relevant accessory cell expresses Ia determinants. Passage of spleen cells over Sephadex G-10 abrogated the response to TNP-Levan and TNP-Dextran as well as to TNP-Ficoll. Addition of adherent accessory cells to the G-10 passed spleen cells reconstituted the response to all 3 antigens. Pretreatment of the adherent accessory cells with a specific anti-Ia serum plus complement abrogated the ability of these cells to provide accessory cell function in the responses to all 3 antigens. Thus, an Ia-positive adherent accessory cell is required for the generation of TI-2 antibody responses to these polysaccharide antigens. This raises the possibility that genetic restrictions may exist between the Ia-positive accessory cell and the lymphocytes involved in the responses to TNP-Ficoll, TNP-Dextran, and TNP-Levan.

Unique T cell Ia antigen expressed on a hybrid cell line producing antigen-specific augmenting T cell factor.

Hybrid cell lines were established by fusion between keyhole limpet hemocyanin(KLH) binding T cells of A/J mice and an AKR T cell tumor line, BW5147. Hybrids were selected for the presence of Ia antigen and KLH-specific augmenting activity of their extracts in the secondary antibody response. The detailed phenotypic and functional analysis of 1 of these clones, FL10, is reported here. The hybrid was positive for both Thy1.1 and Thy1.2 antigens and possessed the Lyt-1+,2-,3- phenotype. Both VH and Ia determinants were detected on their cell surface. The IA locus was mapped in the I-A subregion, but the Ia specificities were serologically distinct from those of B cell Ia antigen. This was demonstrated by the fact that anti-Ia antiserum preabsorbed with B cells could react with the hybrid cells, whereas none of the monoclonal anti-Ia specific for private and public determinations of Iak could. The extract from the cell line specifically augmented the in vitro secondary antibody response against dinitrophenylated KLH, and this activity was removed by absorption with antigen and conventional anti-Ia antisera. The results indicate that the cell line, FL10, carries Ia antigen unique to the T cell, which is associated with the antigen-specific augmenting molecule.

Generation of cytotoxic T lymphocytes by the H-2-encoded E molecules.

Primary CML was generated in strain combinations 4R anti-2R, R107 anti-3R, 7R anti-9R, and GD anti-R101-combinations differing only in the chromosomal interval between the I-A subregion and the Ss locus. No CML could be obtained in any of the reciprocal combinations of these strains. This unidirectionality of the CML reaction correlates with the expression or nonexpression of the E molecules encoded by this interval: the reaction occurred in combinations in which the responder strain lacked and the stimulator strain expressed the E molecules in the cell membrane. The CML reaction was positive when tested on LPS-stimulated blast cells but weak on Con A-stimulated blasts and negative on Ia-negative tumor cells. The reaction could partially be inhibited by monoclonal antibodies to the Ia.m7 determinant presumably carried gy the E alpha chain; it was not inhibited by monoclonal antibodies specific for Ia determinants carried by the A molecule. Cytotoxic lymphocytes specific for a particular combination of E beta and E alpha chains reacted with all cells expressing the particular E beta chain, no matter what the origin of the E alpha chain associated with the E beta chain was. Attempts to generate cytotoxic lymphocytes specifically reactive with allotypic determinants on E alpha chains failed. In F1 hybrids expressing one type of E alpha chain and two types of E beta chain, the single E alpha chain was found to associate with both beta chains, producing two types of E molecule. We conclude from these experiments that the CML determinants detected in the strain combinations used are encoded by the same loci as those coding for the serologically detectable Ia determinants. The CML determinants are carried by the E beta chains; the E alpha chain does not contribute in any way to the specificity of determinant recognition by the cytotoxic lymphocytes. No evidence for allotypic variation of the E alpha chain as detected by the CML assay could be found in this study.

Immunologic effects of whole-body ultraviolet (uv) irradiation: III. Defective splenic adherent cell function in alloantigen-stimulated T-Cell proliferation

The daily exposure of a mouse to ultraviolet (uv) radiation causes a selective depletion of Ia-bearing adherent cells in that animal's spleen. This depletion manifests itself in functional deficiencies in the presentation of protein antigens and haptens to T cells. The present studies demonstrate a defect in splenic adherent cells (SAC) from uv-irradiated mice resulting in defective alloantigen presentation. We show that unfractionated splenocytes and SAC from uv-irradiated mice show decreased stimulatory activity in allogeneic MLR. We then utilize this phenomenon induced by uv radiation to characterize the stimulator cell in the M locus (Mls) determinant-driven MLR. We show that the stimulator cell in Mls determinant-driven MLR is an adherent cell and demonstrate that this stimulator cell bears Ia determinants by showing that whole spleen cells and SAC from mice treated with uv radiation are inefficient stimulators of the Mls determinant-driven MLR. The importance of the Ia determinant on the stimulator cells in Mls determinant-driven MLR is corroborated by the demonstration that a monoclonal antibody directed at this determinant fully blocks the Mls determinant-driven MLR. The significance of these studies to the problem of alloreactions in vivo is discussed.

Acquisition of syngeneic I-A determinants by T cells proliferating in response to poly (Glu60Ala30Tyr10).

The T cell proliferative response in mice to the synthetic polymer GAT is under Ir gene control, mapping to the I-A subregion of the H-2 major histocompatibility complex (MHC). Antigen-dependent proliferation in vitro of in vivo GAT-primed lymph node cells can be inhibited by a monoclonal antibody to Ia-17, an I-A public determinant. Using this antibody for direct immunofluorescent analysis, T cells in GAT-stimulated proliferative culture are identified that express syngeneic I-A during culture. This expression is strictly antigen dependent, requires restimulation in vitro, and requires the presence of I-A-positive adherent antigen-presenting cells. T cells bearing I-A can be enriched by a simple affinity procedure, and I-A-positive cells separated on a FACS are shown to retain antigen-specific reactivity. The acquisition of I-A determinants by T cells under these culture conditions is not nonspecific. The Ia determinants borne by T cell blasts appear to be dictated by the I subregion to which the relevant Ir gene maps, and which codes for the Ia molecule involved in presentation of the antigen. Thus, (B6A)F1 (H-2b X H-2a)F1 LNC express I-Ak antigens when proliferating to GAT but not when stimulated by GLPhe, the response to which is under I-E subregion control. The relation of Ir gene function to Ia-restricted antigen presentation and self-Ia recognition is discussed.

Prolongation of murine islet allograft survival by pretreatment of islets with antibody directed to Ia determinants.

Islets of Langerhans treated with donor-specific anti-Ia serum and complement were transplanted across a major histocompatibility barrier into nonimmunosuppressed diabetic mice. The allografts survived in all recipients for at least 200 days after transplantation. Rejection of an established allograft could be induced by intravenous injection of donor splenocytes. This demonstrates that allografts can serve as targets for immune rejection and supports the possible role of Ia-positive passenger lymphoid cells in initiation of immune rejection. The results show that immunosuppression of the recipient is not a prerequisite for successful transplantation.

Analysis of Ir gene function using monoclonal antibodies: independent regulation of GAT and GLPhe T cell responses by I-A and I-E subregion products on a single accessory cell population.

Abstract T cell proliferative responses to the synthetic polypeptides GAT and GLPhe are under Ir gene control. GAT responses are regulated by gene(s) in the I-A subregion, and GLPhe responses are controlled by a pair of complementing genes mapping to the I-A and I-E subregions. We demonstrate that monoclonal antibody to the I-A gene product inhibits GAT proliferation but not the GLPhe response, whereas a monoclonal antibody to the I-E associated Ia-7 determinant inhibits GLPhe but not GAT proliferation, which indicates independent involvement of each Ia determinant in antigen presentation for the T cell response to these antigens. Use of the same subregion-specific monoclonal antibodies in complement-dependent lysis demonstrates that the antigen-presenting cells for GAT and GLPhe express both I-A and I-E products. The possibility that an Ia subregion-specific "self-receptor" functions on the reactive T cells as a regulatory element is discussed.

Ia antigens on antigen-presenting cells do not carry the same Ia specificities as detected on suppressor and helper T cells.

The passage of spleen cells through a tightly packed nylon wool column partially separated two populations of cells required for the antigen-induced secondary proliferative response of T cells. The proliferating T cells were in the nylon wool column-passed cell population, but the addition of a small number of nylon-wool-adherent cells was required for their maximal proliferative response. Such adherent cells were non-T, non-B, Ia antigen-positive cells serving as antigen-presenting cells. Ia determinants on this cell type are encoded in two separate I subregions, I-A and I-E/C, and they are expressed simultaneously on the same cells. Antisera specific for I-J subregion gene products having as a known activity the killing of antigen-specific suppressor and helper T cells were unable to kill antigen-presenting adherent cells. The results indicated that at least I-J subregion gene products expressed on suppressor and helper T cells are not present on antigen-presenting accessory cells. The Ia specificities detected on accessory cells follow the cross-reactive pattern found in Ia molecules of B cells, and thus antigen-presenting cells carry Ia antigen identical to those of B cells, and not those of T cells.

The Lyt phenotype of a long-term allospecific T cell line. Both helper and killer activities to IA are mediated by Ly-1 cells.

The relationship between surface antigen expression and function in the long-term allospecific T cell line C.C3.11.75 was examined by flow microfluorometry, antiserum plus complement depletion and cell sorting. T cells of the line expressed Lyt-1, but little or no Lyt-2 antigens. Proliferation to cells bearing Iak determinants, generation of T cell-replacing nonspecific helper factor in response to Iak-positive cells and the killing of Iak-positive targets were dependent only on Ly-1 cells. No obvious heterogeneity was found in this cell line despite its disparate functional activities. The fact that Lyt-2 molecules need not be present for killing directed against Ia determinants indicates that such molecules are not obligatory for the induction or delivery of killing and raises the question of what the role of Lyt molecules in T cell recognition or function might be.

Inhibition of antigen-induced proliferation of T cells from radiation-induced bone marrow chimeras by a monoclonal antibody directed against an Ia determinant on the antigen-presenting cell.

Chimeric B10.A T cells that had matured in a (B10.A X B10.Q)F1 environment acquired the ability to respond to poly(Glu56Lys35Phe9) (GL pi), an antigen to which the B10.A mouse is a nonresponder. The response of the chimeric B10.A T cells was initiated by GL phi on responder B10.Q antigen-presenting cells (APC) but not by GL phi on nonresponder B10.A APC. Similarly, chimeric B10.Q T cells that had matured in a (B10.A X B10.Q)F1 environment acquired the ability to respond to poly(Glu60Ala30Tyr10) (GAT) when the antigen was presented on responder B10.A APC, but not when GAT was presented on nonresponder B10.Q APC. No syngeneic haplotype preference was observed for either antigen. These interactions between H-2 nonidentical T cells and APC were inhibited by anti-H-2 antisera and a monoclonal anti-Ia antibody directed against the APC but not by such antibodies when they were directed against the T cell. These data suggest that, when they develop in a responder chimeric environment, genotypic nonresponder T cells become responders by acquiring receptors that allow them to recognize responder I region products on the surface of APC. Furthermore, the data demonstrate that the site of action of the blocking effects of the anti-Ia antibodies is the APC, thus providing strong evidence in support of the idea that Ia antigens on APC are the Ir gene products.

In vitro analysis of allogeneic lymphocyte interaction. V. Identification and characterization of two components of allogeneic effect factor, one of which displays H-2-restricted helper activity and the other, T cell-growth factor activity.

An allogeneic effect factor (AEF) derived from mixed lymphocyte reaction (MLR) cultures of alloactivated A.SW (H-2s) responder T cells and irradiated A/WySn (H-2a) stimulator spleen cells helps an in vitro primary anti-erythrocyte plaque-forming cell PFC response of BALB/c nude spleen cels and also A/WySn but not A.SW T cell-depleted spleen cells. AEF activity is adsorbed by anti-Ik and anti-I-Ak but not by anti-I-Jk, anti-I-ECk, and anti-Is. Gel filtration of ACA 54 resolves AEF into two main components that which appear in the 50,000- to 70,000-mol wt (component I) and 30,000- to 35,000-mol wt (component II) regions, respectively. Component I has a mol wt of 68,000, elutes from DEAE-Sephacel at 0.05-0.1 M NaCl, and has an isoelectric point (pI) of 5.8. It helps A/WySn but not A.SW B cells and, therefore, is H-2 restricted. Component II is not H-2 restricted, because it helps both A.SW and A/WySn B cells. It also stimulates (a) the growth of a long-term cytotoxic cell line in vitro, (b) Con A-induced thymocyte mitogenesis, and (c) the generation of cytotoxic T cells. The latter three properties of component II are not shared by component I. In addition, component II elutes from DEAE-Sephacel at 0.15-0.2 M NaCl and has a pI of 4.3 and 4.9. Ia determinants and Ig VH, CH, L-chain, and idiotypic determinants are not present on either component I or component II. The properties of component II are identical to that of a T cell growth factor produced by Con A-stimulated spleen cells. It is suggested that the H-2-restricted component I of AEF might be an MLR-activated responder T cell-derived Ia alloantigen receptor.

Interleukin 2, production in the syngeneic mixed lymphocyte reaction.

In the syngeneic mixed lymphocyte reaction, Ly 1 cells proliferate following stimulation with Ia-positive macrophage-like cells. The proliferation is accompanied by the release of interleukin 2 into the medium. Both the proliferation and interleukin 2 production are dependent on the expression of Ia antigenic determinants, as the addition of haplotype-specific anti-Ia antisera to the cultures inhibits both parameters.

Stereological meaning of the inflection point count

SUMMARYThe relationship between the inflection point count on a representative microsection and the integral‐asymptotoic line curvature of saddle surface in the system constitutes a new fundamental stereological equation. While the geometric meaning of this quantity cannot be readily visualized, it is unambiguous, and general for all structures. Combinations of the inflection point count with other descriptors of two‐dimensional structures provide potentially useful descriptors of particle roughness, scale, and some aspects of shape. Like all measures of boundary properties, the inflection point count is sensitive to resolution; it is thus essential to report the level of resolution in conjuction with any experimental determination of IA.

Hybrid (combinatorial) Ia specificities: gene complementations to generate Ia.22.

Ia specificity 22 is expressed on a hybrid I-E molecule formed by the association of a beta chain (Ae) coded for by the I-A subregion and an alpha chain (E alpha) encoded by the I-E subregion. Ia.22 can be generated by the complementation of Ab, Ak, As, Ar, with Ed, Ek, Ep, Er, Ew3, Eu, Ev but Eb, Ef, Eq, and Es. With the exception of H-2p which does not complement with As to generate Ia.22, all Ia. 7-positive (I-E) haplotypes can provide the permissive E alpha allele. It is postulated that Ia.22 is a combinatorial Ia determinant generated by the association of the alpha and beta chains. These determinants are probably involved in the immune recognition of antigens under dual Ir-gene control.

Immunochemical characterization of Syrian hamster major histocompatibility complex homologues.

Based primarily on studies in mice and man, the organization and gene-product structures of the mammalian major histocompatibility complex (MHC) are thought to be extensively conserved. However, attempts to generalize from the specific observations of other species to Syrian hamsters have not been completely successful. Previous studies in hamsters have suggested abnormal structure, expression, and/or function of the putative hamster MHC and its products. Characterization of hamster MHC gene-products is therefore of interest. This study concerns the identification and characterization of hamster cell-surface glycoproteins homologous to MHC products of man and mouse. Utilizing radioimmunoprecipitation and serologic techniques, these molecules have been characterized with regard to molecular weight, tissue distribution and immunochemical homology to human and murine class I and II MHC products. In addition, alloantisera raised between histoincompatible hamster strains have been similarly used to identify cell-surface alloantigens of this species. The alloantisera detect cell-surface hamster molecules with immunochemical properties and tissue distribution resembling MHC class II rather than class I products. Thus, in contrast to other species, hamsters appear not to express extensively polymorphic major transplantation antigens of the class I type. Some hamster alloantigens are apparently homologous of Ia determinants since their genes are linked to Hm-1. However, other alloantigens with similar molecular weights are seemingly encoded by genes unlinked to the hamster MHC. These data support the hypothesis that the hamster MHC contains genes which encode for molecular products similar to those described in man and mouse, but that the organization and/or expression of these genes may be atypical.

Major histocompatibility complex-restricted self recognition. A monoclonal anti-I-Ak reagent blocks helper T cell recognition of self major histocompatibility complex determinants.

The functional role of cell surface Ia antigens has been studied for in vitro antibody responses, using as a probe the ability of anti-Ia reagents to inhibit these responses. A hybridoma monoclonal anti-Ia reagent specific for a product of I-Ak (Ia.17) profoundly inhibited in vitro antibody responses to TNP-KLH by spleen cells of the I-Ak but not I-Ab haplotype. This inhibition by anti-I-Ak product, but not by interaction with T or B cell product, in spite of the fact that functional B cells as well as accessory cells could be shown to express the determinant detected by this hybridoma reagent. These results suggest that the Ia expressed by accessory cells in of unique functional importance in these responses. To further characterize the function of Ia antigens in this response system, the mechanism of anti-I-Ak inhibition was determined. The inhibition resulting from interaction of anti-I-Ak with accessory cell Ia was not mediated by nonspecific suppressor cells, nor was there nonspecific interference with accessory cell function as a result of the binding of anti-Ia antibody. The relationship between anti-Ia inhibition and T helper cell recognition of self determinations on accessory cells was analyzed using T cells from radiation bone marrow chimeras. It was demonstrated that (B10 X B10.A)F1 leads to B10 (F1 leads to B10) chimera T cells were able to cooperate with B10 (H-2b and I-Ab) but not B10.A (H-2a and I-Ak) accessory cells for responses to TNP-KLH; F1 leads to B10.A T cells were able to cooperate with B10.A but not B10 accessory cells; and both chimera populations were able to cooperate with (B10 X B10.A)F1 (F1) accessory cells. Monoclonal anti-I-Ak inhibited the cooperation of F1 leads to B10.A T cells with the same F1 accessory cells. Thus, inhibition by anti-I-Ak is dependent upon active helper T cell recognition of I-Ak-encoded determinants expressed on accessory cells. These findings demonstrate that T cells recognize self Ia determinants expressed on accessory cells, and that such recognition is required for the generation of T cell-dependent antibody responses.

Public B cell alloantigen determinants (Ia) and active enhancement of renal allografts in the rat.

Experimental renal transplantation in the rat model has established that immunity to certain donor B cell Ia antigenic determinants is associated with enhanced graft survival. Preimmunization of LEW hosts with BUF lymphoid cells produced significant prolongation (P < 0.005 by log rank analysis) of survival of renal allografts taken from a third-party (LEW X BN)F1. Serological analysis demonstrated that BN and BUF share and MHC-encoded public Ia specificity, expressed on B cells, which we have called Ia.15. Hyperimmune sera directed at this specificity are cytotoxic to 20 +/- 3% of lymph node cells, significantly inhibit the formation of EA rosettes by Fc receptor-bearing cells (EAI), and inhibit mixed lymphocyte reaction (MLR) by 50 to 60% when directed at the stimulator but not when directed at the responder strain cells. Study of a large number of hyperimmune sera raised between inbred rat strains, by EAI and complement-dependent cytotoxicity (CDC) versus B cells, has revealed broad patterns of cross-reactivity, suggesting that public Ia determinants are both complex and highly immunogenic. Immunity restricted to a public Ia antigen plays a role in allograft enhancement.