Ig-free light chains play a crucial role in murine mast cell-dependent colitis and are associated with human inflammatory bowel diseases.

Traditionally, mast cells were regarded as key cells orchestrating type I hypersensitivity responses. However, it is now recognized that mast cells are widely involved in nonallergic (non-IgE) chronic diseases. Also, in inflammatory bowel disease (IBD), a disease not associated with increased IgE concentrations, clear signs of activation of mast cells have been found. In this study, we investigated if Ig-free L chain-induced hypersensitivity-like responses through activation of mast cells could contribute to the pathophysiology of IBD. As a mast cell-dependent model for IBD, mice were skin-sensitized with dinitrofluorobenzene followed by intrarectal application of the hapten. In this murine IBD model, F991 prevented mast cell activation and also abrogated the development of diarrhea, cellular infiltration, and colonic lymphoid follicle hyperplasia. Furthermore, passive immunization with Ag-specific Ig-free L chains (IgLCs) and subsequent rectal hapten challenge elicited local mast cell activation and increased vascular permeability in the colon of mice. Clinical support is provided by the observation that serum concentrations of IgLCs of patients suffering from Crohn's disease are greatly increased. Moreover, increased presence of IgLCs was evident in tissue specimens from colon and ileum tissue of patients with IBD. Our data suggest that IgLCs may play a role in the pathogenesis of IBD, which provides novel therapeutic means to prevent or ameliorate the adverse gastrointestinal manifestations of IBD.

Residual structures in the acid-unfolded states of Vlambda6 proteins affect amyloid fibrillation.

Many proteins form amyloid-like fibrils in vitro under partially or highly unfolding conditions. Recently, we showed that the residual structure in highly unfolded state is closely related to amyloid fibril formation in hen lysozyme. Thus, to better understand the role of the residual structure on amyloid fibril formation, we focused on AL amyloidosis, which results from the extracellular deposition of monoclonal immunoglobulin light-chain variable domains (V(L)s) as insoluble fibrils. We examined the relationship between the residual structure and amyloid fibril formation on three lambda6 recombinant V(L) (rVlambda6) proteins, wild type, Jto, and Wil. Although rVlambda6 proteins are highly unfolded in pH 2, (15)N NMR transverse relaxation experiments revealed nonrandom structures in regions, which include some hydrophobic residues and a single disulfide bond, indicating the existence of residual structure in rVlambda6 proteins. However, the residual structure of Wil was markedly disrupted compared with those of the other proteins, despite there being no significant differences in amino acid sequences. Fibrillation experiments revealed that Wil had a longer lag time for fibril formation than the others. When the single disulfide bond was reduced and alkylated, the residual structure was largely disrupted and fibril formation was delayed in all three rVlambda6 proteins. It was suggested that the residual structure in highly unfolded state has a crucial role in amyloid fibril formation in many proteins, even pathogenic ones.

Prolactin alters the mechanisms of B cell tolerance induction.

OBJECTIVE: Autoimmune diseases predominantly affect women, suggesting that female sex hormones may play a role in the pathogenesis of such diseases. We have previously shown that persistent mild-to-moderate elevations in serum prolactin levels induce a break in self tolerance in mice with a BALB/c genetic background. The aim of this study was to evaluate the effects of hyperprolactinemia on the mechanisms of B cell tolerance induction. METHODS: Effects of prolactin on splenic B cell subsets were studied in female BALB/c mice. B cell receptor (BCR)-mediated apoptosis and proliferation of transitional B cells were analyzed by flow cytometry. Expression of apoptotic genes was examined by microarrays and real-time polymerase chain reaction analysis. B cells coexpressing kappa/lambda light chains were assessed by flow cytometry and immunohistochemistry. Activation status of transitional type 3 (T3) B cells was evaluated by BCR-induced calcium influx studies. RESULTS: BCR-mediated apoptosis of the T1 B cell subset, a major checkpoint for negative selection of autoreactive specificities, was decreased in prolactin-treated mice. Microarray studies indicated that this event may be mediated by the prolactin-induced up-regulation of the antiapoptotic gene interferon-gamma receptor type II and down-regulation of the proapoptotic gene Trp63. Prolactin treatment also altered the amount of receptor editing, as indicated by the increased number of transitional B cells coexpressing kappa/lambda light chains. Additionally, hyperprolactinemia modified the level of B cell anergy by increasing the degree of BCR-induced calcium influx in the T3 B cells. CONCLUSION: Persistently elevated serum prolactin levels interfere with B cell tolerance induction by impairing BCR-mediated clonal deletion, deregulating receptor editing, and decreasing the threshold for activation of anergic B cells, thereby promoting autoreactivity.

Multiple levels of selection responsive to immunoglobulin light chain and heavy chain structures impede the development of Dmu-expressing B cells.

The truncated/V(H)-less mouse H chain Dmu forms precursor B cell receptors with the surrogate L chain complex that promotes allelic exclusion but not other aspects of pre-B cell development, causing most progenitor B cells expressing this H chain to be eliminated at the pre-B cell checkpoint. However, there is evidence that Dmu-lambda1 complexes can be made and are positively selected during fetal life but cannot sustain adult B lymphopoiesis. How surrogate and conventional L chains interpret Dmu's unusual structure and how that affects signaling outcome are unclear. Using nonlymphoid and primary mouse B cells, we show that secretion-competent lambda1 L chains could associate with both full-length H chains and Dmu, whereas secretion-incompetent lambda1 L chains could only do so with full-length H chains. In contrast, Dmu could not form receptors with a panel of kappa L chains irrespective of their secretion properties. This was due to an incompatibility of Dmu with the kappa-joining and constant regions. Finally, the Dmu-lambda1 receptor was less active than the full-length mouse mu-lambda1 receptor in promoting growth under conditions of limiting IL-7. Thus, multiple receptor-dependent mechanisms operating at all stages of B cell development limit the contribution of B cells with Dmu H chain alleles to the repertoire.

Interaction of murine precursor B cell receptor with stroma cells is controlled by the unique tail of lambda 5 and stroma cell-associated heparan sulfate.

Efficient clonal expansion of early precursor B (pre-B) cells requires signals delivered by an Ig-like integral membrane complex, the so-called pre-B cell receptor (pre-BCR). A pre-BCR consists of two membrane micro H chains, two covalently associated surrogate L chains, and the heterodimeric signaling transducer Igalphabeta. In contrast to a conventional Ig L chain, the surrogate L chain is a heterodimer composed of the invariant polypeptides VpreB and lambda5. Although it is still unclear how pre-BCR signals are initiated, two recent findings support a ligand-dependent initiation of pre-BCR signals: 1) a pre-BCR/galectin-1 interaction is required to induce phosphorylation of Igalphabeta in a human precursor B line, and 2) soluble murine as well as human pre-BCR molecules bind to stroma and other adherent cells. In this study, we show that efficient binding of a soluble murine pre-BCR to stroma cells requires the non-Ig-like unique tail of lambda5. Surprisingly however, a murine pre-BCR, in contrast to its human counterpart, does not interact with galectin-1, as revealed by lactose blocking, RNA interference, and immunoprecipitation assays. Finally, the binding of a murine pre-BCR to stroma cells can be blocked either with heparin or by pretreatment of stroma cells with heparitinase or a sulfation inhibitor. Hence, efficient binding of a murine pre-BCR to stroma cells requires the unique tail of lambda5 and stroma cell-associated heparan sulfate. These findings not only identified heparan sulfate as potential pre-BCR ligands, but will also facilitate the development of appropriate animal models to determine whether a pre-BCR/heparan sulfate interaction is involved in early B cell maturation.

Immunoglobulin light chains dictate vesicular transport-dependent and -independent routes for IgM degradation by the ubiquitin-proteasome pathway.

Degradation of IgM mu heavy chains in light chain-negative pre-B cells is independent of vesicular transport, as is evident by its insensitivity to brefeldin A or cell permeabilization. Conversely, by the same criteria, degradation of the secretory mu heavy chain in light chain-expressing B cells depends on vesicular transport. To investigate whether the presence of conventional light chains or the developmental stage of the B-lymphocytes dictates the degradative route taken by mu, we express in 70Z/3 pre-B cells either lambda ectopically or kappa by lipopolysaccharides-stimulated differentiation into B cells and show their assembly with mu heavy chains. The resulting sensitivity of mu degradation to brefeldin A and cell permeabilization demonstrates that conventional light chains, a hallmark of B cell differentiation, are necessary and sufficient to divert mu from a vesicular transport-independent to a vesicular transport-dependent degradative route. Although both routes converge at the ubiquitin-proteasome degradation pathway, only in light chain-expressing cells is vesicular transport a prerequisite for mu ubiquitination.

A point mutation in the constant region of Ig lambda1 prevents normal B cell development due to defective BCR signaling.

Surface expression of B cell antigen receptors (BCRs) containing Ig and Igalpha/Igbeta generates signals required to transit discrete developmental checkpoints. The mechanism by which BCR components collaborate to initiate signals is still unclear. The expression of Iglambda1 in SJL mice is 50-fold lower than in other strains. Here, we demonstrate by gene targeting that a point mutation, which changes a glycine to a valine in the lambda1 constant region, is responsible for this defect. In vitro experiments show that Ig receptors bearing this mutation, while expressed normally, are deficient in signaling. These findings reveal a direct involvement of the Ig light chain (IgL) in B cell signaling and development beyond the requirement of light chains for BCR assembly.

Influence of the isotype of the light chain on the properties of IgG.

It is widely appreciated that the isotype of the H chain of the Ab molecule influences its functional properties. We have now investigated the contribution of the isotype of the L chain to the structural and functional properties of the Ab molecule. In these studies, the L chain variable region of a murine anti-dansyl Ab was joined to either human kappa or lambda constant region domains and expressed with mouse-human chimeric H chains of the four human IgG isotypes. The resulting Abs were secreted as fully assembled molecules although, as has been previously observed, IgG4 with either kappa or lambda L chains was also secreted as HL half-molecules. However, the isotype of the L chain can influence the kinetics of intracellular assembly with IgG1lambda, IgG2lambda, and IgG4lambda assembling more slowly than their kappa counterparts. The isotype of the L chain also influenced the susceptibility of the interchain disulfide bonds to attack by reducing agents with variable effects, depending on the isotype of the H chains. For IgG2, but not for IgG1, -3, and -4, the isotype of the L chain influenced the rate of clearance in mice, with IgG2lambda having a shorter in vivo half-life than IgG2kappa. Only slight differences were also observed between lambda and kappa molecules in their kinetics of binding to and dissociation from the hapten dansyl. These studies demonstrate that the isotype of the L chain has only a slight impact on the structural and functional properties of variable region identical Abs.

Conventional and surrogate light chains differentially regulate Ig mu and Dmu heavy chain maturation and surface expression.

Positive selection of precursor (pre-) B cells by Ig membrane mu H chains (mum HC) and counterselection mediated by the truncated HC Dmu depend on the ability of each HC to form a pre-B cell receptor (pre-BCR) signaling complex with the surrogate L chain (SLC) components lambda5 and Vpre-B. To better understand how pre-BCR signaling output is determined by its Ig components and the SLC, we investigated the regulation of pre-BCR surface expression and HC secretory maturation in a new nonlymphoid system. We took this approach as a means to distinguish B-lineage-specific effects from pre-BCR-intrinsic properties that may influence these aspects of pre-BCR homeostasis necessary for signaling. As in pre-B cells, the SLC in nonlymphoid cells supported only a limited degree of mum HC maturation and low pre-BCR surface expression levels compared with conventional LCs, indicating that this was due to an intrinsic property of the SLC. We identified the non-Ig region of lambda5 as harboring the restrictive activity responsible for this phenotype. This property of lambda5 was also evident with Dmu, but the overall SLC- and L chain-dependent requirements for Dmu maturation and surface expression were markedly different from those for mum. Surprisingly, Dmu was modified in an unusual manner that was only dependent on Vpre-B. These results establish a novel function of lambda5 in limiting surface pre-BCR levels and reveal biochemical properties of Ig molecules that may underlie the diverse consequences of pre-BCR signaling in vivo by different HCs.

Cytotoxicity of myeloma light chains in cultured human kidney proximal tubule cells.

We evaluated the effect of eight species of light chains on cultured human kidney proximal tubule cell proliferation. Exposure to light chains for 48 hours caused dose-dependent inhibition in tritium ((3)H)-thymidine incorporation by simian virus 40 immortalized human proximal tubule cells, although the effect was variable among different species of light chains. We studied cytotoxic effects of selected toxic light chains in further detail. Two of these light chains caused significant DNA degradation. A lambda-light chain caused lactate dehydrogenase release from exposed cells at 48 hours, but not at 24 hours. Cytomorphological and electron microscopic examination of cells exposed to light chains for 24 hours showed condensed nuclei, cell detachment, paucity of mitotic activity, and apoptosis, and at 48 hours of exposure, changes consistent with necrosis. Apoptosis assay by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method showed a sixfold increase in the number of apoptotic cells exposed to the same lambda-light chain for 24 hours. Rhodamine-phalloidin staining showed variable but significant disruptions in the actin cytoskeleton. These studies show that some myeloma light chains are toxic to cultured human proximal tubule cells and induce cytoskeletal injury and DNA damage consistent with apoptosis followed by secondary necrosis. Direct proximal tubule cell toxicity may be an important mechanism of renal involvement in multiple myeloma.

Inhibition of intracellular transport of B cell antigen receptor complexes by Kaposi's sarcoma-associated herpesvirus K1.

The B cell antigen receptor (BCR) is a large complex that consists of a disulfide-linked tetramer of two transmembrane heavy (mu) chains and two light (lambda or kappa) chains in association with a heterodimer of Igalpha and Igbeta. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a transforming protein called K1, which has structural and functional similarity to Igalpha and Igbeta. We demonstrate that K1 downregulates the expression of BCR complexes on the surface. The NH(2)-terminal region of K1 specifically interacts with the mu chains of BCR complexes, and this interaction retains BCR complexes in the endoplasmic reticulum, preventing their intracellular transport to the cell surface. Thus, KSHV K1 resembles Igalpha and Igbeta in its ability to induce signaling and to interact with mu chains of the BCR. However, unlike Igalpha and Igbeta, which interact with mu chains to direct BCR complexes to the cell surface, K1 interacts with mu chains to block the intracellular transport of BCR complexes to the cell surface. These results demonstrate a unique feature of the K1 transforming protein, which may confer virus-infected cells with a long-term survival advantage.

Nephritogenic lambda light chain dimer: a unique human miniautoantibody against complement factor H.

A unique monoclonal Ig lambda light chain dimer (protein LOI) was isolated from the serum and urine of a patient with hypocomplementemic membranoproliferative glomerulonephritis. In vitro the lambda light chain dimer efficiently activated the alternative pathway of complement (AP). When added to normal human serum, LOI temporarily enhanced AP hemolytic activity, but during a prolonged incubation the hemolytic activity was depleted. Protein LOI was found to bind to factor H, the main regulator molecule of AP. By binding to the short consensus repeat domain 3 of factor H, the dimer LOI blocked one of three interaction sites between H and C3b and thus inhibited the activity of H and induced an uncontrolled activation of the AP. Structural analysis showed that LOI belonged to the Vlambda3a subgroup of lambda light chains. The variable (V) region of LOI was most closely related to the predicted product of the Vlambda3 germline gene Iglv3s2, although it contained several unique residues that in a tertiary homology model structure form an unusual ring of charged residues around a hydrophobic groove in the putative Ag binding site. This site fitted considerably well with a putative binding site in the molecular model of domain 3 of factor H containing a reciprocal ring of charged amino acids around a hydrophobic area. Apparently, functional blocking of factor H by the Ab fragment-like lambda light chain dimer had initiated the development of a severe form of membranoproliferative glomerulonephritis. Thus, the lambda light chain dimer LOI represents the first described pathogenic miniautoantibody in human disease.

Control of the sizes and contents of precursor B cell repertoires in bone marrow.

Ordered rearrangements of immunoglobulin (Ig) gene loci, first as DH to JH, then as VH to DHJH, and finally as VL to JL segment-specific recombinations occur 'in-frame' and 'out-of-frame'. 'In-frame' rearrangements lead to the expression of truncated DHJH-microC proteins and to microH chains. These H chain proteins have two major effects on precursor B cells. They suppress (as DJC mu proteins) or enhance (as full microH chain) the proliferation of precursor cells at the point where these precursors express these proteins. At the same time, they signal allelic exclusion of the microH chain alleles, so that VH to DHJH rearrangement at the second allele is suppressed. Regulation of precursor B cell proliferation and H chain allelic exclusion is mediated by a pre-B cell receptor that is composed of the microH chains and a surrogate L chain. This surrogate L chain is made up of two proteins encoded by the Vpre-B and lambda 5 genes that are expressed only at the early precursor cell stages just before and when H chain genes are first expressed. They are not found in later B cell development, when L chains are expressed, nor in any other cell of the body tested so far. The physiological roles of surrogate L chain and of the pre-B receptor have been clarified by generating mutant mice in which the lambda 5 gene has been inactivated by targeted disruption. Molecular mechanisms and cellular developments, by which the pre-B receptor controls proliferation and allelic exclusion, are discussed.

Ig light chains are secreted predominantly as monomers.

Ig light (L) chains are secreted not only as part of assembled Ab molecules, but also as free L chains, the latter process being involved in the pathology of several diseases. The secretion competence of free L chains distinguishes them from free subunits of other oligomeric proteins, which are usually retained intracellularly. We used several techniques to test the idea that secretion of free L chains is dependent on dimerization. Coexpression of pairs of L chains, differing in only one amino acid, which alters the secretory phenotype, shows that these L chains behave independently: the wild-type chains are secreted, whereas the mutants are retained intracellularly. A survey of kappa- or lambda-producing cell lines by nonreducing gel electrophoresis shows that a negligible fraction of these L chains exists as disulfide-bonded dimers. Moreover, chemical cross-linking and density gradient centrifugation demonstrate that there is no significant pool of noncovalent L chain dimers. Noncovalent heterodimers can be detected readily between a kappa-chain and a chimera consisting of a heavy chain variable domain linked to the kappa-chain constant domain. This confirms that noncovalent L chain homodimers would have been detected if they were present. These findings about the association state of free L chains are independent of the host cell, as they are observed in both myeloma cells and COS fibroblasts. We conclude that L chain dimerization is a rare event that neither facilitates secretion nor is required for it.

Cloning and analysis of IgG kappa and IgG lambda anti-thyroglobulin autoantibodies from a patient with Hashimoto's thyroiditis: evidence for in vivo antigen-driven repertoire selection.

Antibodies to thyroglobulin (Tg) are commonly found in patients with the autoimmune thyroid diseases Graves' disease and Hashimoto's thyroiditis as well as in individuals with apparently normal thyroid function. Although it is not clear how Tg Abs are involved in the pathology of the diseases, the study and analysis of these Abs may nevertheless be instructive in allowing the development of an Ab response to an autoimmune disease-associated self Ag to be followed. We have prepared IgG kappa and lambda phage display combinatorial libraries from the cervical lymph node of a single Hashimoto's thyroiditis patient with a high anti-Tg titer. These were selected with purified human Tg, and 10 IgG kappa and 9 IgG lambda clones were analyzed further. Sequence analysis of the clones showed a very highly restricted heavy chain usage and a less restricted light chain usage. There was a variable degree of divergence from germ-line sequence in the light chain sequences, with a clear relationship between relatively higher affinity of the Fab for human Tg and an increased degree of somatic hypermutation. The Tg-selected Fab did not bind to Tg from other species, to reduced denatured Tg, or to thyroid peroxidase. The Fab inhibited patient serum binding to human Tg by between 39 and 79%. In summary, we have isolated 19 high affinity, human Tg-specific Fab and shown that the relative affinity of the Fab is directly related to the pattern of somatic hypermutation.

The lambda B cell repertoire of kappa-deficient mice.

Analysis of the B cell repertoire is complicated by the huge diversity inherent in the germ line determined combinatory. Making use of knockout technology, kappa-deficient mice have been obtained. They constitute a shrewd model to follow the expression of an Ig minilocus, such as the lambda one, in the normal condition compared with classical transgenic models. Indeed, in contrast to wild type mice, in which only 5% of lambda B cells are produced, these mutant mice exclusively produce lambda positive B cells. Although, the lambda locus is well characterized and has a relatively simple organization, the mechanistic and selective pressures that govern its utilization are still poorly understood. The analysis of the lambda B cell repertoire in kappa-deficient mice, should therefore bring more conclusive informations. Here we present the lambda subtype distribution in the various cellular compartments of the kappa-deficient mice, and discuss the rules that can be responsible for this distribution. Our recent data indicate that the lambda subtype proportions in the bone marrow and the spleen result, for the major part, from mechanistic processes (i.e., recombinase accessibility, production of V-J functional joint and H/L pairings) while the lambda proportions found in the peritoneal cavity ensue from selective processes. Finally, the capacity to respond to various antigens is discussed from such a generated lambda B cell repertoire.

lambda 5, but not mu, is required for B cell maturation in a unique gamma 2b transgenic mouse line.

gamma 2b transgenic mice have a severe B cell defect, apparently caused by strong feedback inhibition of endogenous H-gene rearrangement coupled with an inability of gamma 2b to provide the survival/maturation functions of mu. A unique gamma 2b transgenic line, named the C line, was found to permit B cell development. When the C line is crossed with a mu-membrane knockout line, gamma 2b+ B cells develop in the homozygous knockout. In contrast, a transgenic line representative of all the other gamma 2b lines is completely B cell deficient when mu-mem is deleted. Strikingly, the C phenotype is dominant in C x other gamma 2b transgenic line crosses. There is no evidence for higher gamma 2b transgene expression or other position effects on the transgene in the C mouse. The sequences of the three gamma 2b transgene copies in the C line are identical to that of the original transgene. These results have led to the conclusion that in the C line the transgene integration constitutively induces a gene whose expression can replace mu. To more clearly delineate the stage at which the altered phenotype of the C line is expressed, C mice were crossed onto a lambda 5 knockout background. In the absence of lambda 5, the C line produces no B cells. Since it was also found that gamma 2b can associate with the surrogate light chain (sL; lambda 5/Vpre-B), the crosses between C line gamma 2b mice and lambda 5 knockout mice suggest that gamma 2b/sL is required for B cell maturation in this mouse line. Thus, gamma 2b alone is unable to replace mu for pre-B cell survival/maturation; however, in combination with an unknown factor and the sL, gamma 2b can provide these nurturing functions.

Immunologic memory to phosphocholine keyhole limpet hemocyanin. Recurrent mutations in the lambda 1 light chain increase affinity for antigen.

Anti-phosphocholine (PC)-keyhole limpet hemacyanin hybridomas representative of a memory response that express the lambda 1 L chain isotype have a high reactivity to PC-protein. A common feature of these hybridomas possessing high affinity for PC-protein is the occurrence of somatic mutations resulting in replacement changes in three CDR2 positions of the lambda 1 L chain. The influence of each of these three positions on the Ag binding properties of these antibodies was examined by site-specific mutagenesis and expression of recombinant antibody molecules by transfected cells. Affinity measurements and fine specificity profile determinations demonstrated the importance of the three lambda 1 CDR2 positions in Ag binding. Compared to antibodies expressing germline lambda 1, including one with an additional junctional serine that is not encoded by V or J, those antibodies possessing critical changes in CDR2 would have a strong selective advantage based on affinity differences for Ag. Sequence analysis of a group of clonally related hybridomas expressing mutated lambda 1 genes allowed construction of a hypothetical genealogic tree that suggests selection based on changes in CDR2 of lambda 1 in the absence of H chain mutations. The results are consistent with stepwise acquisition of mutations and selection based on affinity constraints.

Gene transfer of immunoglobulin light chain restores heavy chain secretion.

Several lines of evidence suggest that immunoglobulin (Ig) light (L) chain plays a role in the secretion of heavy (H) chain. For example, myeloma variant lines, which synthesize the Ig H chain but not the L chain, fail to secrete H chain protein. Here we have tested directly the role of chain assembly in the control of Ig secretion by the transfer of functional L chain genes into two such L chain-defective myeloma mutants. A lambda 2 or kappa L chain gene was introduced into variant lines of the mouse myelomas MOPC 315 (IgA, lambda 2) or PC7 (IgM, kappa), respectively. Although the two mutant lines are unable to secrete the H chain they produce, rescue of secretion of complete Ig protein molecules (IgA or IgM) was observed after transfection. These results imply that the secretory apparatus of these cells is intact and that the failure to secrete free H chain reflects a structural feature intrinsic to that protein. The implications of these results with respect to control of secretion of multi-subunit proteins are discussed.

Determinant-specific regulation of T helper cell responses to murine lambda light chains by both H-2 and non-H-2 genes.

Previous work has revealed that the T helper cell (Th) responses to an antigenic determinant of V lambda 2(315) (called lambda 2.1) is regulated by both H-2 and non-H-2 genes. In the present study this was confirmed and extended to two other determinants, one shared between free lambda 2(315) and lambda 1(J558) (called lambda 2.2) and one unique for free lambda 1(J558) (called lambda 1.1). H-2 genes regulate the responses to the latter determinants, because BALB.B (H-2b) mice were low responders and BALB/c (H-2d) mice were high responders. Thus, the H-2d haplotype on BALB/c background was associated with high responder status. However, when the H-2d haplotype was examined on other genetic backgrounds than BALB/c, the animals could be classified as either intermediate or low responders, depending upon the non-H-2 background. This demonstrated that non-H-2 genes also influenced Th responses. Furthermore, C3H-H-2o, DBA/2 and B10.D2 mice (all H-2d) responded to only one (lambda 2.1) but not the other (lambda 2.2) of two determinants physically linked on the same polypeptide chain (lambda 2(315)). This indicated that the non-H-2 gene effect is capable of fine discrimination, i.e. the non-H-2 gene-mediated low responder phenotype may at least in part be due to failure of recognition of certain antigenic sites, like the H-2-linked Ir-gene defect. F1 hybrids responded to the same determinants as their parental strains; e.g., the BALB/c non-H-2 background exerted a dominant influence over the low responder background of C3H, B10 and DBA/2 strains.