Ek beta mutant antigen-presenting cell lines expressing altered Ak alpha molecules

Antigen-presenting cells (APC) expressing mutant Ek beta and Ak alpha proteins were isolated after chemical mutagenesis of TA3 cells and negative immunoselection for altered Ek beta molecules. Mutant clones were analyzed for biosynthesis, assembly, and cell surface expression of altered Ia molecules, and were assayed for antigen-presenting function by using a variety of T cell clones. Three types of mutants were detected: type 1, which had lost expression of the Ek beta chain and produced altered Ak alpha chains; type 2, which also expressed altered Ak alpha chains, and which expressed Ek beta proteins that had lost reactivity to the 17.3.3 and 74D monoclonal antibodies (mAb), but retained reactivity to other anti-Ek beta mAb; and type 3, which had lost expression of both Ek beta and Ak beta: Ak alpha surface molecules. Thus, all of the mutant clones that produced modified Ak alpha proteins also displayed either total loss or serologic modification of the Ek beta molecule. Ek beta:E alpha-reactive T cell clones were not stimulated when type 1 or type 3 cells were used as APC, but all such T cells were fully reactive with type 2 mutant APC. Most Ak beta:Ak alpha-reactive T cell clones could respond to type 1 and 2 APC, and none were responsive to type 3 APC. However, two autoreactive Ak beta:Ak alpha-specific T cell hybridomas were stimulated only very weakly by type 1 and type 2 cells expressing modified Ak alpha proteins. These results demonstrate that Ia mutations can have highly selective effects on antigen presentation to T cells as well as on mAb binding, and thus suggest that individual Ia molecules may be composed of many different functional subsites.     

Separation of the immune response genes for LDH-B and MOPC-173. II. Description of an immune response defect in B10.ASR7

By using the intra-I region recombinant mouse strain B10.ASR7 (H-2as3), the immune response (Ir) genes for LDH-B and MOPC-173 were genetically and serologically separated, as assayed by T cell proliferation. Previous work demonstrated that H-2s and H-2b strains respond to LDH-B and MOPC-173 whereas H-2a and H-2k strains failed to respond due to haplotype-specific suppression of I-Ak molecule-activated T helper cells by I-Ek molecule-activated T suppressor cells. In the experiments reported here, B10.ASR7 mice, which lack I-Ek expression, mounted a significant T cell proliferative response to LDH-B but not to MOPC-173. Separation of the Ia determinants used in restricting these two antigen responses was further confirmed when pretreatment of B10.S(9R) (A beta sA alpha sE beta sE alpha k) macrophages with A.TL anti-B10.HTT serum (anti-As beta Es beta Js) adsorbed with B10.ASR7 spleen cells blocked the MOPC-173 response but not the LDH-B response. Unadsorbed serum blocked both antigen responses. The B10.ASR7 E beta allele was determined to be s due to the ability of (A.TL X B10.ASR7)F1 hybrids to mount a T cell proliferative response to the terpolymer GLPhe. Monoclonal antibody blocking of the B10.ASR7 T cell proliferative response to LDH-B demonstrated that the Ia.2 and Ia.17, and not the Ia.15 epitopes are spatially related to the Ia epitopes involved in the restriction of the B10.ASR7 LDH-B T cell proliferative response. In addition, B10.ASR7 helper T cells generated in response to LDH-B were suppressed in a haplotype-specific manner by I-Ek molecule-restricted suppressor T cells in that (A.TL X B10.ASR7)F1 hybrids failed to respond to LDH-B. This nonresponsiveness was eliminated by treatment with monoclonal antibodies directed against the I-Ek molecule. These results suggest the possibility that the immune response defect in B10.ASR7 could be related to the site of recombination.     

Clonal analysis of B and T cell responses to Ia antigens. IV. Proliferative T cell clones recognizing E beta and/or E alpha allodeterminants

The allospecific T cell recognition of the I-Ek molecule was assessed by using eight A. TH anti-A. TL proliferative T cell clones, all of which expressed the Thy-1-2+, Lyt-1+, Lyt-2-, Ia-, and p94,180+ cell surface phenotype. The use of panels of stimulating cells from homozygous of F1 hybrid strains indicated each T cell clone exhibited specificity for distinct alloactivating determinants including: i) a private E beta k-controlled determinant expressed in cis- or trans-complementing E beta kE alpha strains; ii) an apparently nonpolymorphic E alpha determinant resembling the serologic specificity Ia.7, i.e., present in all strains carrying E alpha and E beta expressor alleles; and iii) a series of conformational I-E determinants, the expression of which required a precisely defined combinatorial association of E beta plus E alpha chains. Two clones were found to be reactivated by cis- but not trans-complementing E beta k E alpha k strains, and another recognized an allodeterminant shared by the I-Ab molecule. Various I-Ek-reactive monoclonal antibodies (mAb) directed to epitopes presumably expressed on either E alpha (epitope clusters I and II) or E beta (epitope cluster III) chains inhibited the proliferative responses of seven clones recognizing private E beta k or unique E beta E alpha conformational activating determinants. By contrast, the restimulation of the clone directed to a nonpolymorphic E alpha determinant was selectively blocked by anti-Ia.7 mAb defining epitopes on the E alpha chains but not by those directed to the E beta chain. On the basis of these data, it was concluded that the recognition sites of most anti-I-Ek proliferative T cells were expressed on the E beta chain or the E beta plus E alpha interaction products, and that a minority of such alloreactive T cells could be activated through recognition of the E alpha chain per se.     

Functionally distinct agretopic and epitopic sites. Analysis of the dominant T cell determinant of moth and pigeon cytochromes c with the use of synthetic peptide antigens

The dominant T cell determinant on moth and pigeon cytochromes c in B10.A (E beta k:E alpha k) mice is located in the C-terminal portion of the protein, contained within residues 93-103 or 93-104. Thirty-seven antigen analogs, containing single amino acid substitutions at positions 98, 99, 101, 102, 103, and 104, were synthesized. The effects of the substitutions on in vitro antigenicity and in vivo immunogenicity were determined. Functional assays with T cell clones identified residues 99, 101, 102, and 103 as critical, based on their effect on antigenic potency. Peptides containing substitutions at residues 99, 101, and 102 were capable of eliciting unique clones upon immunization of B10.A mice. This was consistent with the identification of these residues as part of the epitope, the site on the antigen that interacts with the T cell receptor. Immunization with peptides substituted at residue 103, however, failed to elicit clones with unique specificity for the immunogen. When these peptides were tested for their ability to stimulate the T cell clones with antigen-presenting cells from B10.A(5R) mice expressing the E beta b:E alpha k Ia molecule, a consistent change in the relative antigenic potency was observed with 50% of the peptides. The effect of the Ia molecule on the antigenic potency ruled out the possibility that residue 103 nonspecifically affected antigen uptake or processing and identified residue 103 as part of the agretope, the site that interacts with the Ia molecule. The locations of the agretope and the epitope on this antigenic determinant appear to be fixed, even in the presence of large numbers of amino acid substitutions. However, some substitutions were found to affect both the agretope and the epitope, placing limits on the functional independence of the two sites. The results are discussed in terms of the trimolecular complex model of T cell activation and the implications of these data for antigen-Ia molecule interactions.     

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.     

Structure-function analysis of Ia molecules: in-phase insertion mutagenesis of the amino-terminal domain of the E beta k polypeptide chain

To identify which segments of the beta 1 domain of the E beta k polypeptide control T cell recognition of antigen, E beta genes were constructed with in-phase insertion mutations. Five independent mutants, with insertions mapping to positions 24, 50 and 93 of the E beta k polypeptide, were obtained. Cell lines expressing these mutated genes were analysed by microfluorometry using a panel of 20 anti-Ek monoclonal antibodies. None of the tested in-phase insertions has resulted in the loss of antibody binding sites. In striking contrast, mutations at position 93 and at a lesser level 50 were indicative of a crucial role of the corresponding regions in T-cell recognition, because they led to significant or complete loss of antigen-presenting function with all but one of the T hybridomas tested. These data are discussed with regard to a model of the foreign antigen binding site of Ia molecules.     

IA mutant functional antigen-presenting cell lines

We describe a protocol for the selection of mutant cells with an altered pattern of Ia antigenic determinants and antigen-presenting properties from a homogeneous population of functional antigen-presenting cells (APC). The APC line used in this work was obtained by fusing lipopolysaccharide-stimulated B cells from (BALB/c x A/J)F1 donors with cells from the M12.4.1 BALB/c B lymphoma cell line. The resulting hybridomas, including TA3, retained the potent antigen-presenting activity of the parental B lymphoma line and expressed Ia antigens and immune response gene-determined antigen-presenting properties of the A/J type. Mutants of TA3 were obtained by subjecting the cells to negative immunoselection with one monoclonal anti-(alpha) 1-Ak antibody and complement followed by positive immunoselection via electronic cell sorting with a second monoclonal alpha I-Ak or alpha I-Ek antibody. Two types of mutants were obtained. One, A8, appeared to have undergone a fairly limited alteration, since it lost only some of the I-Ak antigenic determinants; the second type appeared to have lost the entire I-Ak molecule but to have retained the I-E molecule. Functional studies with the A8 mutant demonstrated that the loss of a limited number of I-Ak determinants correlated with the loss of a specific I-Ak-encoded restriction element, since A8 failed to present a specific antigen, hen egg lysozyme (HEL), to a HEL-specific I-Ak-restricted T cell hybridoma but retained some capacity to present a second antigen, poly(Glu60Ala30Tyr10) (GAT), to a GAT-specific I-Ak-restricted T cell hybridoma. These results indicate that Ia antigens are the products of immune response gene loci. The availability of such mutants should allow an examination of the relationship between the structure of an Ia molecule and the antigens with which it is co-recognized by T cells.     

The Ia.2 epitope defines a subset of lipid raft-resident MHC class II molecules crucial to effective antigen presentation

Previous work established that binding of the 11-5.2 anti-I-A(k) mAb, which recognizes the Ia.2 epitope on I-A(k) class II molecules, elicits MHC class II signaling, whereas binding of two other anti-I-A(k) mAbs that recognize the Ia.17 epitope fail to elicit signaling. Using a biochemical approach, we establish that the Ia.2 epitope recognized by the widely used 11-5.2 mAb defines a subset of cell surface I-A(k) molecules predominantly found within membrane lipid rafts. Functional studies demonstrate that the Ia.2-bearing subset of I-A(k) class II molecules is critically necessary for effective B cell-T cell interactions, especially at low Ag doses, a finding consistent with published studies on the role of raft-resident class II molecules in CD4 T cell activation. Interestingly, B cells expressing recombinant I-A(k) class II molecules possessing a β-chain-tethered hen egg lysosome peptide lack the Ia.2 epitope and fail to partition into lipid rafts. Moreover, cells expressing Ia.2(-) tethered peptide-class II molecules are severely impaired in their ability to present both tethered peptide or peptide derived from exogenous Ag to CD4 T cells. These results establish the Ia.2 epitope as defining a lipid raft-resident MHC class II conformer vital to the initiation of MHC class II-restricted B cell-T cell interactions.     

Identification of distinct predominant epitopes recognized by myoglobin-specific T cells under the control of different Ir genes and characterization of representative T cell clones

We characterized the fine specificity of antigen recognition of myoglobin-immune T cells from B10.D2, B10.GD, and B10.A(5R) mice. Polyclonal H-2d T cells show a predominant response to an epitope centering on Glu 109 and His 116. These residues localize a dominant epitope to one segment of the myoglobin surface, but probably are not the only amino acid residues involved. This response pattern maps genetically to I-Ad (or Kd) based on the B10.GD recombinant strain. A different epitope specificity was detected in B10.A(5R) T cells mapping to I-E beta b E alpha k or I-Cd, but no difference was detected between strains differing at the Igh locus. Thus, epitope specificity varied with Ia haplotype but not Igh allotype. Myoglobin-specific B10.D2 T cell lines were established, and five clones specific for the Glu 109, His 116 epitope were isolated; these were all restricted to I-Ad antigen-presenting cells. These clones represent the dominant specificity in the polyclonal T cell response to myoglobin and will be useful in characterizing the structure and function of T cell receptors for antigen and Ia. The differences in number and nature of T cell epitopes compared to B cell epitopes of myoglobin are discussed.     

Expression of CD5 regulates responsiveness to IL-1

The role of the CD5 surface molecule in T cell responsiveness to IL-1 was examined. A CD5-mutant Jurkat cell line was generated from a CD5+ parent cell line. This CD5- mutant subclone was infected with a defective retrovirus containing the CD5 cDNA and/or the neo gene encoding G418 resistance. The CD5+ wild type Jurkat produced IL-2 in response to anti-CD3 mAb, OKT3, cross-linked to a solid surface. IL-2 production was enhanced by co-culture with IL-1 or anti-CD5 Mab. Neither the CD5- mutant nor the CD5- G418-resistant infectant responded to anti-CD5 mAb or to IL-1. Responsiveness to IL-1 was restored by cell surface expression of CD5 in the CD5+ infectant. Both the CD5+ wild type Jurkat and the CD5+ infectant responded equivalently to purified IL-1, IL-1 alpha and rIL-1 beta. Optimal concentrations of IL-1 and anti-CD5 mAb had an additive effect on the enhancement of IL-2 production stimulated with cross-linked anti-CD3 mAb suggesting that IL-1 and CD5 act through distinct, complementary pathways to augment T cell activation. The correlation of CD5 expression and specific binding of rIL-1 beta was examined in these cell lines. Both the specific binding (at 4 degrees C) and subsequent internalization (at 37 degrees C) of 125I labeled rIL-1 beta was equivalent in the CD5+ infectant and the CD5+ wild type Jurkat cell, whereas specific binding of 125I-labeled rIL-1 beta was markedly decreased in the CD5-G418-resistant infectant. These observations strongly suggest that cell surface expression of CD5 regulates binding of and responsiveness to IL-1.     

Is a natural ligand of the T lymphocyte CD2 molecule a sulfated carbohydrate?

Recent studies have implicated sulfated polysaccharide (SP) recognition in a range of cell adhesion systems. Inasmuch as the CD2 (E rosette receptor, T11, LFA-2) molecule of human T lymphocytes is a cell surface glycoprotein involved in the adhesion of T cells to various target cells the possibility that CD2 binds SP was investigated. It was found that E rosetting of human T lymphocytes, a phenomenon involving CD2, was readily inhibited by the SP dextran sulfate (DxS) and, to a lesser extent, by the sulfated polymer polyvinyl sulfate whereas 11 other SP had no effect on E rosetting, this effect occurring at the T cell level. mAb binding studies revealed that DxS and polyvinyl sulfate, but none of the other SP tested, inhibited the binding to T cells of the anti-CD2 mAb OKT11 and anti-T112 but augmented expression of the T113 epitope of the CD2 molecule. In contrast, DxS had little or no effect on the binding of anti-CD3, -CD4, -CD8, -Pgp-1 and WT31 (TCR alpha/beta) mAb. Direct evidence that CD2 binds DxS was demonstrated by the ability of DxS-coupled fibers to totally deplete the CD2 Ag from lysates of radiolabeled human T lymphocytes and by the quantitative recovery of the CD2 Ag in fiber eluates. Control fibers coupled with other SP bound little or no CD2. Collectively, the data indicate that the CD2 molecule specifically binds DxS and suggest that a potential target cell ligand for CD2 is a sulfated carbohydrate structure.     

The I-A beta b region (65-85) is a binding site for the superantigen, staphylococcal enterotoxin A

Ia antigen is a receptor for the superantigen staphylococcal enterotoxin A (SEA). Peptides I-A beta b(30-60), I-A beta b(50-70), I-A beta b(65-85), and I-A beta b(80-100) of the MHC class II antigen beta chain on mouse (H-2b) accessory cells were synthesized. Only I-A beta b(65-85) inhibited SEA binding to the mouse B-cell lymphoma line, A20 (H-2d) and the human Burkitt's lymphoma line, Raji (HLA-DR). The I-A beta b(65-85) sequence is a predicted alpha-helix along the hypothetical antigen binding cleft of the Ia molecule. I-A beta b(65-85) also directly and specifically bound both the intact SEA molecule and its Ia binding site, represented by the peptide SEA(1-45). The results suggest that I-A beta b region (65-85) is a necessary site for Ia molecular interaction with the superantigen SEA. Further, the data suggest that the same helical region of other Ia antigens binds SEA irrespective of haplotype and species.     

Separation of the immune response genes for LDH-B and MOPC-173. I. Description of an immune response defect in B10.BASR1

By using the intra-I-region recombinant mouse strain, B10.BASR1 (H-2as4), the immune response (Ir) genes for LDH-B and MOPC-173 were genetically and serologically separated, as assayed by T cell proliferation. Previous work demonstrated that the H-2s and H-2b strains respond to LDH-B and MOPC-173, whereas the H-2a and H-2k strains failed to respond due to haplotype-specific suppression of I-Ak-activated T helper cells by I-Ek-activated T suppressor cells. In the experiments reported here, B10.BASR1 mice, which lack I-Ek expression, mounted a significant T cell proliferative response to MOPC-173 but not to LDH-B. Separation of the Ia determinants used in restricting these two antigen responses was further confirmed when pretreatment of B10.S(9R) (A alpha sA beta sE beta sJk) macrophages with A.TL anti-B10.HTT (anti-A beta sE beta sJs) serum absorbed with B10.BASR1 spleen cells blocked the LDH-B response but not the MOPC-173 response. Unabsorbed serum blocked both antigen responses. The primary immunogenic determinant recognized by LDH-B or MOPC-173 immune T cells was not present on both antigens, as MOPC-173-primed T cells and LDH-B-primed T cells responded only to the priming antigen. Lastly, by using the A beta mutant strain, B6CH-2bm12, it was shown that the Ir gene and Ia determinants affected by this mutation had no effect on the LDH-B and MOPC-173 proliferative responses. These results suggest the possibility of an intragenic recombinatorial event in either the A alpha or A beta chain resulting in the separation of these two immune response gene functions.     

The molecular basis of class II MHC allelic control of T cell responses.

To identify the molecular basis for the effects of MHC molecule polymorphism on T cell responses, we have combined functional T cell response testing with measurements of peptide binding to the class II MHC molecules on transfected cells. Our studies identify a small subset of spatially localized polymorphic residues of the E alpha E beta dimer (strand residue beta 29, and helix residues beta 72 and beta 75) regulating cytochrome c peptide presentation by two distinct mechanisms. The first effect is on quantitative control of net peptide binding. The replacement of the valine found at position beta 29 in E beta k with the glutamic acid found in E beta b results in a selective loss of pigeon cytochrome peptide but not moth cytochrome peptide binding to the resultant mutant E alpha E beta k molecule. Reciprocally, the replacement of glutamic acid at beta 29 in E beta b with valine results in a gain of pigeon peptide binding. These changes in binding parallel changes in T cell responses in vitro to these peptide-E alpha E beta combinations and mirror the in vivo immune response gene phenotypes of mice expressing E alpha E beta k and E alpha E beta b. E alpha E beta s molecules, which have a beta 29 glutamic acid, are nevertheless able to bind and present pigeon cytochrome peptides, and this is due to changes in helix residues beta 72 and beta 75 that compensate for the negative effect of the beta 29 glutamic acid. The second activity is a critical change in the conformation of the peptide bound to the same extent by distinct MHC molecules, as revealed by changes in T cell responses to moth cytochrome peptides presented by two E alpha E beta molecules differing only at position beta 29. Both of these effects can be ascribed to a single polymorphic residue modeled to be inaccessible to TCR contact (beta 29), providing a striking demonstration of how MHC molecule polymorphism can modify T cell-dependent immune responses without direct physical participation in the receptor recognition event.     

Cytofluorometric isolation of I937, an Ia antigen-bearing variant of the Ia-negative human monocytic cell line U937

Cells of the human monocyte cell line U937 are generally considered devoid of any Ia antigens on their surface. In analyzing U937 cells with a large panel of monoclonal anti-human Ia antibodies by flow cytometry, we detected a small number of cells that appeared to react with antibodies to HLA-DR and HLA-DS/DC molecules. These Ia-positive cells were isolated and were cloned, resulting in a human monocyte cell line that expresses high levels of Ia antigens. We analyzed these antigens by one- and two-dimensional polyacrylamide gel electrophoresis, after radiolabeling and immunoprecipitation. Three distinct Ia molecules, alpha 1 beta 1, alpha 1 beta 3 (HLA-DR-like), and alpha 2 beta 2 (HLA-DC/DS-like) are synthesized by I937 cells, alpha 1 beta 3 molecule being the predominant species. The Ia antigen-bearing human monocyte cell line is expected to be useful for studying events involved in antigen presentation.     

Binding of staphylococcal enterotoxin A to HLA-DR on B cell lines

Staphylococcal enterotoxin A (SEA) is a potent polyclonal T cell activator. Its activating effect is entirely dependent upon its binding to accessory cells. Monocytes, B cells, and B lymphomas can bind SEA and support activation of T cells. We have earlier found that Raji cells are particularly efficient as accessory cells for SEA-induced T cell proliferation. In the present investigation we have used this cell line for the isolation and characterization of the membrane molecule to which SEA binds. Flow cytometric analysis of cells dually stained with SEA and anti-HLA-DR mAb showed that the amount of bound SEA was proportional to the HLA-DR expression. Electrophoresis of detergent extracts of Raji cells revealed one distinct SEA-binding band with a Mr of 60 to 65 kDa. This band had the same electrophoretic mobility as the MHC class II molecules. A mAb (G8) with the ability to block SEA binding to Raji cells was established. This mAb was shown to bind to the HLA-DR molecule. Both the G8 mAb and an anti-HLA-DR mAb 9-49 inhibited SEA binding to accessory cells and also inhibited SEA-induced, but not PHA-induced, T cell proliferation and production of IL-2. Immunoprecipitation with specific anti-HLA-DR and anti-HLA-DQ mAb demonstrated that SEA binds to the HLA-DR molecule but not to the HLA-DQ molecule. Binding SEA to Raji cells followed by cross-linking and detergent solubilization of cell membranes, electrophoresis, and Western blotting resulted in two SEA-containing bands corresponding to a Mr of 90 and 105 kDa, respectively. Both these bands also contained the HLA-DR molecule and their appearance could be blocked by preincubation of the Raji cells with the G8 mAb. Collectively the results show that the HLA-DR molecule is the main functional molecule for binding of SEA to accessory cells and that this binding of SEA to HLA-DR is a necessary requirement for SEA-induced T cell activation.     

Influenza viruses as lymphocyte mitogens. II. Role of I-E molecules in B cell mitogenesis by influenza A viruses of the H2 and H6 subtypes

Influenza A viruses of the H2 and H6 subtypes behave as T cell-independent B cell mitogens for lymphocytes from strains of mice that express the class II MHC glycoprotein I-E (Ia.7+ haplotypes). We have examined the role of I-E molecules in mitogenesis by these viruses. Lymphocytes from (Ia.7+ X Ia.7-)F1 hybrid strains that express lower levels of I-E antigen than homozygous Ia.7+ strains showed a level of response to H2 and H6 influenza viruses that was intermediate between the high response of the Ia.7+ parent and the low response of the Ia.7- parent. The mitogenic response of H-2k lymphocytes to these viruses was completely inhibited by low concentrations of anti-I-Ek monoclonal antibody that had no effect on B cell proliferation induced by LPS or by influenza A virus of the H3 subtype. Furthermore, incubation of H-2k spleen cells with high concentrations of H2 (but not H3) influenza viruses substantially inhibited the binding of radio-labeled anti-I-Ek, but not anti-I-Ak, monoclonal antibody. Cell mixing experiments indicated that expression of I-E by the B cells was critical to the mitogenic response, whereas I-E expression by accessory cells may not be necessary. The data support a model in which B cell mitogenesis by these viruses results from direct binding of the viruses to I-E molecules on B lymphocytes.     

A single monoclonal anti-Ia antibody inhibits antigen-specific T cell proliferation controlled by distinct Ir genes mapping in different H-2 I subregions

A xenogeneic rat anti-mouse Ia monoclonal antibody, M5/114 (gamma 2b, kappa), was studied for its effects in vitro on T cell proliferative responses. Strain distribution studies revealed that M5/114 could inhibit I-A subregion-restricted T cell responses of the H-2b,d,q,u but not the H-2f,k,s haplotypes, indicating that this xenoantibody recognizes a polymorphic determinant on mouse Ia molecules. This same monoclonal antibody was found to inhibit BALB/c (H-2d) T cell proliferation to both G60A30T10 and G58L38 phi 4. The Ir genes regulating responses to these antigens map to either the I-A subregion (GAT), or the I-A and I-E subregions (GL phi), raising the possibility that M5/114 recognizes both I-A and I-E subregion-encoded Ia glycoproteins. It could be shown, using appropriate F1 responding cells, that M5/114 does in fact affect GAT and GL phi responses by interaction with both the I-A and the I-E subregion products, and not by any nonspecific effect resulting from binding to the I-A subregion product alone. These results are consistent with genetic and biochemical studies directly demonstrating that M5/114 recognizes A alpha A beta and E alpha E beta molecular complexes. The existence of a shared epitope on I-A and I-E subregion products suggests the possibility that these molecules arose by gene duplication. Finally, the precise correlation between the Ia molecules recognized by M5/114 and the ability of this antibody to block T cell responses under Ir gene control strengthens the hypothesis that Ia antigens are Ir gene products.     

Three classes of signalling molecules on B-cell membranes

The question of whether surface immunoglobulin and Ia molecules have a signalling function in helper T cell-dependent activation of B cells has been evaluated. Two sources of B cells have been used, one a purified population of hapten-binding B cells, the other a B-cell lymphoma, CH12, with known antigen specificity. Evidence is presented that both immunoglobulin and Ia molecules are receptors actively involved in the initial activation of resting B cells. Nevertheless, the requirements for ligand binding to either receptor can be bypassed under appropriate conditions, and the implications of this result for the function of these molecules is discussed. With respect to B-cell Ia, the authors present data that demonstrate two distinct functions of this molecule, one as a restricting element for T-cell activation, the second as a signalling receptor for B-cell excitation. On the CH12 surface, the I-A molecule fulfills the former function, but T-cell interactions with I-A fail to result in B-cell stimulation, suggesting that B-cell Ia may limit helper T cell-B cell interactions. We suggest that the binding of antigen surface immunoglobulin and binding of helper T-cell receptors to the appropriate Ia molecule(s) results in the activation of genes that encode for a third class of membrane B-cell receptors, those that bind B-cell stimulating factors.     

Induction of tyrosine phosphorylation during ICAM-3 and LFA-1-mediated intercellular adhesion, and its regulation by the CD45 tyrosine phosphatase

Intercellular adhesion molecule (ICAM)-3, a recently described counter- receptor for the lymphocyte function-associated antigen (LFA)-1 integrin, appears to play an important role in the initial phase of immune response. We have previously described the involvement of ICAM-3 in the regulation of LFA-1/ICAM-1-dependent cell-cell interaction of T lymphoblasts. In this study, we further investigated the functional role of ICAM-3 in other leukocyte cell-cell interactions as well as the molecular mechanisms regulating these processes. We have found that ICAM-3 is also able to mediate LFA-1/ICAM-1-independent cell aggregation of the leukemic JM T cell line and the LFA-1/CD18-deficient HAFSA B cell line. The ICAM-3-induced cell aggregation of JM and HAFSA cells was not affected by the addition of blocking mAb specific for a number of cell adhesion molecules such as CD1 1a/CD18, ICAM-1 (CD54), CD2, LFA-3 (CD58), very late antigen alpha 4 (CD49d), and very late antigen beta 1 (CD29). Interestingly, some mAb against the leukocyte tyrosine phosphatase CD45 were able to inhibit this interaction. Moreover, they also prevented the aggregation induced on JM T cells by the proaggregatory anti-LFA-1 alpha NKI-L16 mAb. In addition, inhibitors of tyrosine kinase activity also abolished ICAM-3 and LFA-1- mediated cell aggregation. The induction of tyrosine phosphorylation through ICAM-3 and LFA-1 antigens was studied by immunofluorescence, and it was found that tyrosine-phosphorylated proteins were preferentially located at intercellular boundaries upon the induction of cell aggregation by either anti-ICAM-3 or anti-LFA-1 alpha mAb. Western blot analysis revealed that the engagement of ICAM-3 or LFA-1 with activating mAb enhanced tyrosine phosphorylation of polypeptides of 125, 70, and 38 kD on JM cells. This phenomenon was inhibited by preincubation of JM cells with those anti-CD45 mAb that prevented cell aggregation. Altogether these results indicate that CD45 tyrosine phosphatase plays a relevant role in the regulation of both intracellular signaling and cell adhesion induced through ICAM-3 and beta 2 integrins.