DNA-mediated transfer of major histocompatibility class II I-Ab and I-Abm12 genes into B lymphoma cells: molecular and functional analysis of introduced antigens

A20.2J B lymphoma cells have been co-transfected with the A alpha b, A beta b or with the A alpha b, A beta bm12 and neomycin resistance genes. The transfected cell lines constitutively express the I-Ab or I-Abm12 class II molecules at a level comparable with that of the endogenous I-Ad antigen. The I-Ab antigens expressed on three independently transfected B cell clones (A20.Ab.1, A20.Ab.2, and A20.Ab.3) are serologically and functionally indistinguishable from the I-Ab molecules expressed by control H-2bxd B hybridoma cells (LB cells). These transfected cell lines were potent I region-restricted antigen-presenting cells to a large panel of antigen-specific, autoreactive and alloreactive T cell hybridomas, as well as normal T cell clones. There were not significant differences in the efficiency of antigen presentation by the Ia molecules encoded by the transfected, as compared with the endogenous, I-A genes. The expression of a functional I-Ab antigen on the surface of cells transfected with A beta bm12 and A alpha b genes is consistent with previous work that implicated the A beta-chain alone in the bm 12 mutation. Furthermore, because the transfected A20.Ab and A20.Abm12 cells display the serologic and functional properties of normal spleen cells from the wild-type and mutant mouse strains, respectively, it is clear that class II genes do not undergo unexpected and unpredictable alterations after transfection in this system. This system permits us to investigate the structural requirements for interactions between class II major histocompatibility complex antigens, a foreign antigen, and the T cell receptor by in vitro site-directed mutagenesis coupled with DNA-mediated gene transfer.     

The Eb beta gene may have acted as the donor gene in a gene conversion-like event generating the Abm 12 beta mutant

At least two different class II histocompatibility antigens, I-A and I-E, are encoded by the murine major histocompatibility complex. Both types of class II antigens are composed of polypeptide chains called alpha and beta. Class II antigens display extensive genetic polymorphism, the main part of which resides in the NH2-terminal domains of the A alpha, A beta and E beta chains. Recently it was shown that the mutant gene Abm 12 beta differed from the wild-type gene Ab beta by three nucleotide substitutions, which all occur within a stretch of 14 nucleotides. Multiple substitutions of the type found in the Abm 12 beta gene suggest that the mutant arose by a gene conversion-like event. To examine whether the Eb beta gene may have served as the donor gene in the generation of the Abm 12 beta gene, we have isolated and sequenced a cDNA clone corresponding to the Eb beta gene. Comparisons of the Eb beta, the Ab beta and the Abm 12 beta nucleotide sequences revealed that the Eb beta sequence is identical to that of Abm 12 beta in the positions where the latter differs from the Ab beta sequence. This observation is consistent with the notion that the Abm 12 beta mutant gene arose by a gene conversion-like event involving the Eb beta gene.     

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.     

Ia specificities on parental and hybrid cells of an I-A mutant mouse strain

Splenic B cells and B cell blasts from the I-A mutant mouse strain B6.C-H-2bm12 were tested by serology with a series of new monoclonal anti-Iab antibodies. Four out of 5 of those monoclonal antibody-defined specificities that are determined by wild-type I-Ab antigens were undetectable on B6.C-H-2bm12 cells. Specificities both present and absent on mutant cells appear to be determinants on the same wild-type molecule, as indicated by sequential precipitation experiments with soluble H-2b antigens. The lack of expression of certain Ia specificities on mutant cells was found not to be the result of disparate control by the Xid gene, which was previously shown to control the expression of Ia.W39, another specificity absent in B6.C-H-2bm12 mice. Serologic testing of Ia specificities on cells and blasts from F1-hybrid mice suggested that the Iabm12 antigens are codominantly expressed, indicating a failure to detect trans regulation or complementation of the mutant phenotype. Another monoclonal antibody-defined Ia specificity dependent on the expression of the E beta polypeptide was normally expressed in B6.C-H-2bm12 mice. These data thus suggest that the lesion of these mutant mice occurred in the A alpha and/or A beta structural gene, resulting in the loss of several Ia specificities.     

The structure-function relationship of I-A molecules: a biochemical analysis of I-A polypeptides from mutant antigen-presenting cells and evidence of preferential association of allelic forms

Chemically induced mutants of an I-Ak,d-expressing, antigen-presenting B cell-B lymphoma hybridoma have recently been generated by immunoselection in vitro with I-Ak-specific monoclonal antibodies, and were found to possess alterations in some of the I-Ak region-dependent functions. The mutants were categorized as alpha-polypeptide mutants or beta-polypeptide mutants on the basis of the patterns of reactivity with anti I-Ak alpha and anti I-Ak beta monoclonal antibodies. To delineate the structural alterations underlying the differences in serologic and functional properties of these mutants, I-A molecules from several of these mutant hybridomas were compared biochemically with wild type I-Ak polypeptides by two-dimensional gel electrophoresis and high-pressure liquid chromatographic (HPLC) tryptic peptide analyses. These results suggest that the marked alterations in antibody reactivity and T cell-activating functions of the beta-polypeptide mutants G1, K2, and LD3, as well as the Ia alpha-polypeptide mutant JE50, may be due to very limited alterations in the Ia polypeptides. The functional deficiencies of the alpha-polypeptide mutant JE67 could be attributed to the change in net charge exhibited by its Ak alpha polypeptide. HPLC tryptic peptide analysis of I-A molecules isolated from the alpha-polypeptide mutant J4 indicates that the functional deficiencies exhibited by this mutant are due to a complete loss of expression of the Ak alpha polypeptide. The inability to detect significant amounts of Ad alpha Ak beta and Ak alpha Ad beta hybrid molecules in immunoprecipitates from some of these cell lines suggests that some hybrid molecules may be expressed at low levels due to preferential Ia polypeptide chain association. Together, these results indicate that most serologically defined epitopes are localized on either one or the other Ia polypeptide, whereas T cell-defined epitopes are determined by a combination of both Ia polypeptides. The results of these analyses also enable us to evaluate different immunoselection strategies for the most efficient production of mutants expressing limited alterations in Ia polypeptides.     

The structure-function relationship of I-A molecules: correlation of serologic and functional phenotypes of four I-Ak mutant cell lines

We have isolated and characterized four mutant I-Ak-expressing cell lines derived from the B cell-B lymphoma hybrid antigen-presenting cell line TA3. The mutants were isolated by first selecting against expression of one Ak epitope by treatment with a monoclonal antibody in the presence of complement and then selecting for retention of a second Ak epitope by electronic cell-sorting of cells stained for fluorescence with a second monoclonal antibody. The serologic and functional phenotypes of the mutants were characterized by using panels of I-Ak-specific monoclonal antibodies and I-Ak-restricted T hybridomas. We obtained one Ak alpha mutant (J4) that no longer reacts with any Ak alpha-specific antibody and also is incapable of stimulating any I-Ak-restricted T hybridoma. We obtained three Ak beta mutants (LD3, K5, G1) that express a wide range of serologic and functional phenotypes. Correlation of the serologic and functional phenotypes reveals that the serologic epitope Ia.1 may overlap with a major site of T cell recognition, whereas the Ia.17 serologic epitope appears to be only a minor site for T cell recognition.     

Functional sites on the A alpha-chain. Polymorphic residues involved in antigen presentation to insulin-specific, Ab alpha:Ak beta-restricted T cells

The interaction between the clonally selected TCR, the processed Ag peptide and the Ia molecule is not fully understood in molecular terms. Our study intended to delineate the residues of Ab alpha molecules that function as contact sites for Ag and for the TCR of a panel of T cells specific for the A chain of insulin in combination with mixed haplotype Ab alpha:Ak beta molecules. Multiple L cell transfectants expressing alpha,beta-heterodimers composed of wild-type A beta- and chimeric or mutant A alpha-chains served as antigen presenting cells. The recombinant A alpha-chains had been generated by an exchange of allelically hypervariable regions (ahv) or amino acids. The results point out a broad spectrum of b sequence requirements for the bovine insulin-specific activation of the various T cell populations. Activation of some T cells seemed quite permissive, requiring b-haplotype amino acids in any one of the three ahv, while others had strict requirements, demanding b-haplotype sequence in all three ahv. Our data stress the role of ahvII and especially ahvIII in T cell activation. Interestingly, single amino-acid substitutions in ahvII or ahvIII of Ak alpha were sufficient to bring up full stimulation potential for two T cell hybridomas. We also found that some ahv permutations influenced the Ag preference (beef insulin versus pig insulin) of some T cells. These data suggest a critical role for the three-dimensional structure of the complex formed by Ia and the processed Ag peptide. The stability of the trimolecular complex essential for T cell activation is envisioned as being the sum of the interactions between Ag/I-A, TCR/Ag, and TCR/I-A, each variable in strength and compensated for by the others.     

Delineation of Ia:nominal antigen complementary determinants recognized by T cells in studies of gene complementation in response to insulin

The immune response to beef insulin in mice is controlled by genes in the IA subregion. We have previously shown that B6.C-H-2bm12 (bm12) mice, an A beta gene mutation of B6, have a selective loss of responsiveness to beef insulin, whereas other IAb controlled responses such as (TG)AL and collagen are unchanged. F1 hybrid mice between two nonresponder genotypes Ik and Ibm12 were found to be good responders to beef insulin suggesting functional complementation. In this report, we define the cellular and molecular basis of this complementation by investigating the determinants on Ia molecules and nominal antigen that are recognized by (B10.A X bm12)F1 proliferating T cells. Genetic analyses demonstrated that the Ik region was the only nonresponder genotype that complemented Ibm12, thus restoring responsiveness to beef insulin. More precisely an IAk and not an IEk gene product was found to be responsible for this complementation. Antibody blocking studies furthermore showed that the A alpha b:A beta k hybrid Ia mediated the response to beef insulin in (B10.A X bm12)F1 mice. Clonal analyses of the response to beef insulin in these F1 mice confirmed these conclusions, because the insulin-specific response in all 21 F1-T cell clones studied thus far was found to be dependent upon presentation via the A alpha b:A beta k hybrid Ia molecule. Dissection of the antigenic specificity of the F1-T cell clones demonstrated recognition of at least two insulin determinants, one A-loop (A8-A10) associated and the other non-loop (A4 or B chain) associated. Therefore these studies identify the molecular and antigenic basis of the Ir gene complementation seen in the response to beef insulin of (B10.A X bm12)F1 hybrids.     

Molecular analysis of antigen recognition by insulin-specific T-cell hybridomas from B6 wild-type and bm12 mutant mice.

Molecular analysis of the heterodimeric T-cell antigen receptor of insulin-specific class II-restricted T-cell hybridomas (THys) derived from C57BL/6 (B6) wild-type and B6.C-H-2bm12 (bm12) mutant mice revealed that such T cells use a diverse V gene repertoire. Analysis of three THys that use related V genes, however, showed a number of novel features. Two THys that share major histocompatibility complex restriction use V alpha genes that are 98.6% homologous. Two THys sharing the same antigen fine specificity use a particular germ line V beta D beta J beta combination. A 21-base-pair deletion in the 5' segment of the J beta gene occurs in one THy, suggesting a novel mechanism for generating diversity in T-cell antigen receptor beta genes. The first amino acid encoded by N sequences at the V-D junction is conserved in a pair of T cells which recognize identical antigenic epitopes. The implications of these findings for the structural mechanisms underlying major histocompatibility complex-restricted antigen-specific T-cell recognition are discussed.     

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.     

T cell responses to select Ia determinants using the I-A mutant mouse strain B6.C-H-2bm12

The T cell repertoire of B6.C-H-2bm12 mice (an I-A mutant mouse strain) to wild-type Iab antigens was investigated using both secondary proliferative cultures and cloned T cell lines. Because bm12 mice have a gain-loss mutation of their gene encoding the Ia beta-chain polypeptide, bm12 anti-B6 T cell responses are specific for the select component of Iab specificities that was lost as a result of the mutation. Although stimulator cells bearing Iab antigens elicited the strongest responses, Iaq, d, and s antigens also resulted in reproducible stimulations of these bm12 anti-B6-primed T cells. Cloned T cell lines isolated from bm12 anti-b6 cultures revealed similar findings, with most clones recognizing determinants unique for Iab antigens; however, clones showing cross-reactions with Iad and/or q were also selected. Using F1 hybrid responder T cells (mutant x cross-reactive strain), we further dissected this cross-reactivity into several distinct cross-reactive determinants. Because bm12 mice lack the serologically defined Ia differentiation antigen W39, T cell recognition of this determinant was investigated by using bm12 anti-B6-primed cells. Stimulation by Ia.W39+ cells was appreciably better than by Ia.W39- (Xid-defective) cells, suggesting that bm12 T cells recognize an Xid-regulated, W39-like Ia differentiation antigen.     

Complex regulation of class II gene expression: analysis with class II mutant cell lines

Several Ia-negative variants of a homozygous Iad-expressing antigen-presenting B lymphoma cell line, M12, have been obtained by repeated cycles of negative immunoselection after mutagenesis with ethylmethane sulfonate or gamma-irradiation. Two such Iad-negative cell lines, selected with a mixture of alpha I-Ad and alpha I-Ed monoclonal antibodies, failed to present antigen to all cloned Iad-restricted T cells tested, whereas the third cell line, selected with alpha I-Ad reagents only, stimulated I-Ed but not I-Ad-restricted T cells. The mutations in all three cell lines resulted in the absence of RNA specific for the A beta d gene. In addition, two-dimensional gel electrophoresis of immunoprecipitates from one of the I-Ed-negative cell lines demonstrated the presence of intracytoplasmic Ed polypeptides that exhibited significantly decreased amounts of oligosaccharide-induced heterogeneity. The introduction of class II A beta b and A alpha b genes by DNA-mediated transfection resulted in the serologic and functional expression of a class II I-Ab molecule but not the reexpression of the endogenous class II molecules; thus a transacting regulatory element is unlikely to be the target of the mutagenic event. The analysis of these and other Ia variant cell lines may prove useful in understanding the molecular mechanisms that control the expression of class II molecules in B cells.     

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.     

Receptor diversity of insulin-specific T cell lines from C57BL (H-2b) mice

To characterize the T cell receptor repertoire in an immune response in which the Ia and nominal antigenic determinants are defined and limited, we have cloned and sequenced the expressed receptors from four independent, beef insulin-specific T cell lines from C57BL mice. Each of these lines responded to beef but not to the pork insulin, thus defining the nominal antigenic determinant recognized. Furthermore, each of these lines could only be presented antigen by B6 but not mutant B6.C-H-2bm12 antigen-presenting cells, thus defining the requisite Ia recognition or antigen-association site. In spite of this functional similarity in ligand specificity, each of these T cell lines was found to use different V alpha and V beta gene segments. Moreover, structural comparisons of implied protein sequences of each of these receptors showed no stretches of conserved amino acid residues that could be implicated in ligand interaction. However, the V alpha genes used by these four clones appeared considerably more homologous to each other than were their V beta genes.     

Idiotypic and fluorometric analysis of the antibodies that distinguish the lesion of the I-A mutant B6.C-H-2bm12

The serologic lesion of the I-A mutant mouse strain, bm12, was investigated with the use of monoclonal anti-Iab antibodies and anti-idiotypic (Id) reagents produced against these antibodies. In a fluorometric analysis, three different monoclonal anti-Iab antibodies (25-9-17, 34-5-3, 28-16-8) failed to bind bm12 cells, whereas two anti-Iab antibodies (25-5-16 and 17/227), which bound bm12 cells, showed about one-half the fluorescence intensity that they showed in binding to Iab antigens. Of the three monoclonal antibodies that failed to react with bm12 cells, two antibodies (25-9-17 and 34-5-3) were found to bind the same steric site on Iab molecules (cluster I). In contrast, the antibodies (25-5-16 and 17/227) that reacted with both Iab and Iabm12 antigens were found to bind a second distinct site (cluster II). The binding of antibody 28-16-8 to Iab antigens inhibited reciprocally the binding of cluster I and II anti-Iab antibodies, suggesting a possible third site, sterically located intermediate between the other two sites. To assess the relatedness of the antibodies defining the serologic lesion of bm12 mice, xenogeneic and syngeneic anti-Id reagents were produced against antibodies 25-9-17 and 28-16-8. By using these anti-Ids in a binding site-related inhibition assay, a cross-reactive idiotype was detected that is shared by 25-9-17 and 34-5-3 antibodies; thus these two monoclonal antibodies share several features, including 1) idiotypic determinants, 2) failure to bind bm12 cells, 3) binding the same spatial Iab site, and 4) having indistinguishable serologic fine specificity that corresponds with a previously defined predominant alloantigenic determinant recognized in the bm12 anti-Iab humoral response. Therefore, several parameters of antibody recognition of Ia can now be correlated with structural changes in Ia molecules. These findings will potentiate future studies of the T cell recognition of these same Ia epitopes.     

In vivo treatment with monoclonal anti-I-A antibodies: disappearance of splenic antigen-presenting cell function concomitant with modulation of splenic cell surface I-A and I-E antigens

A single injection of anti-I-Ak antibody (AB) into H-2k mice resulted in abrogation of splenic antigen-presenting cell (APC) function for protein antigen-primed T cells or alloantigen-specific T cells. Spleen cells from anti-I-A-treated mice are not inhibitory in cell mixing experiments when using cloned antigen-specific T cells as indicator cells, thus excluding a role for suppressor cells in the observed defect. Also, nonspecific toxic effects and carry-over of blocking Ab were excluded as causes for the defect. Experiments with anti-I-Ak Ab in (H-2b X H-2k)F1 mice showed abrogation of APC function for T cells specific for both parental I-A haplotypes. In homozygous H-2k mice, anti-I-Ak treatment not only abrogated APC function for I-Ak-restricted cloned T cells but also for I-AekE alpha k-restricted cloned T cells. FACS analysis of spleen cells from anti-I-Ak-treated (H-2b X H-2k)F1 mice revealed the disappearance of all Ia antigens (both I-A and I-E determined), whereas the number of IgM-bearing cells was unaffected. The reappearance of APC function with time after injection was correlated with the reappearance of I-A and I-E antigen expression. In vitro incubation of spleen cells from anti-I-A-treated mice led to the reappearance of Ia antigen expression and APC function within 8 hr. Thus, it appears that B cells (as determined by FACS analysis) and APC (as determined by functional analysis) behave similarly in response to in vivo anti-I-A Ab treatment. We interpret these findings as suggesting that in vivo anti-I-A treatment temporarily reduces the expression of Ia molecules through co-modulation on all Ia-bearing spleen cells, thereby rendering them incompetent as APC. Such modulation of Ia molecules does not occur when spleen cells are incubated in vitro with anti-I-A antibodies. These results imply that a primary defect purely at the level of APC in anti-I-A-treated mice may be responsible for the observed T cell nonresponsiveness when such mice are subsequently primed with antigen.     

Structural comparison of I-A antigens produced by a cloned murine T suppressor cell line with B-Cell-Derived I-A

A cloned, antigen-specific T suppressor cell line derived from a CBA mouse expresses large amounts of I-A and I-E antigens. Comparative two-dimensional polyacrylamid gel electrophoresis of biosynthetically labeled I-A antigens immunoprecipitated with a variety of monoclonal I-Ak-specific antibodies suggested that alpha, beta and Ii polypeptide chains are identical with B-cell-derived I-A. Dimeric complexes formed by I-A chains derived from B or T suppressor cells were also similar with two major exceptions. Pulse-labeled T-cell-derived Ia antigen was complexed with two additional unknown components of about 31K. These components were not visible in pulse-chased (processed) materials. In addition, T suppressor-cell-derived I-A antigens did not contain S-S linked dimers consisting of processed alpha and beta chains, which are usually formed during solubilization of B cells. We consider the possibility that in T cells these chains are associated with other structures, thus preventing S-S linkage between alpha and beta chains.     

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.     

In vivo treatment with anti-I-A antibodies: differential effects on Ia antigens and antigen-presenting cell function of spleen cells and epidermal Langerhans cells

The in vivo activation of T cells by a variety of antigens can be inhibited by the administration of anti-I-A antibodies (Ab) at the time of antigen priming. This inhibition can partially be explained by the temporary loss of Ia molecules from Ia-bearing antigen-presenting cells (APC) in the spleen. In this study, the effects of i.p. injected monoclonal Ab specific for I-A glycoproteins of different H-2 haplotypes on Ia antigen expression and APC function of spleen cells and epidermal Langerhans cells were compared. It was found that anti-I-A Ab quickly bound to both spleen cell and Langerhans cell Ia antigens. Although spleen cell Ia antigens were modulated and thus temporarily disappeared, Ia antigen expression by epidermal Langerhans cells was not modulated. In functional studies, the capacity of spleen cells and epidermal cells from anti-I-A Ab treated vs control animals to function as APC for antigen-specific, I-A- or I-E-restricted T cell clones was tested. A single injection of anti-I-A Ab completely abolished the APC function of spleen cells as shown in several inbred mouse strains, F1 animals, and with the use of several different Ab and T cell clones. In contrast, Langerhans cell-dependent APC function of epidermal cells remained completely unaltered. Even multiple injections of high doses of Ab never caused any inhibition of Langerhans cell function. Experiments with anti-I-Ak or anti-I-Ad Ab in an (H-2k X H-2d)F1 animal showed abrogation of APC function of spleen cells, but again not of Langerhans cells. Thus in vivo anti-I-A Ab administration appears to differentially affect Ia antigen expression and APC function from spleen and epidermis: Ia antigens are modulated from spleen cells but not from epidermis, and APC function disappears in the spleen but not in the epidermis. The abrogation of splenic but not of Langerhans cell APC function with anti-I-A Ab will facilitate the dissection of the relative contributions of Langerhans cells as compared with other APC in the generation of cutaneous immune responses.     

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.