Kappa-deficient Mice Research Articles

Increased dietary cholesterol promotes enhanced mutagenesis in DNA polymerase kappa-deficient mice

DNA polymerase kappa (Polκ) bypasses planar polycyclic N2-guanine adducts in an error-free manner. Cholesterol derivatives may interact with DNA to form similarly bulky lesions. In accordance, these studies examined whether increased mutagenesis of DNA accompanies hypercholesterolemia in Polk-/- mice. These mice also carried apoE gene knockouts to ensure increased levels of plasma cholesterol following exposure to a high cholesterol diet. The mice carried a reporter transgene (the λ-phage cII gene) for subsequent quantitative analysis of mutagenesis in various tissues. We observed significantly increased mutation frequencies in several organs of apoE-/-Polk-/- mice following a high cholesterol diet, compared to those remaining on a standard diet. Regardless of dietary regime, the mutation frequency in many organs was significantly higher in apoE-/-Polk-/- than in apoE-/-Polk+/+ mice. As expected for polycyclic guanine adducts, the mutations mainly consisted of G:C transversions. The life expectancy of apoE-/-Polk-/- mice maintained on a high cholesterol diet was reduced compared to apoE-/-Polk+/+ mice. Overall, this study demonstrates a role for Polκ in bypass of cholesterol-induced guanine lesions.

DNA polymerase kappa deficiency does not affect somatic hypermutation in mice.

Somatic hypermutation (SH) in B cells undergoing T cell-dependent immune responses generates high-affinity antibodies that provide protective immunity. Most current models of SH postulate the introduction of a nick into the DNA and subsequent replication-independent, error-prone short-patch synthesis by one or more DNA polymerases. The Pol kappa (DinB1) gene encodes a specialized mammalian DNA polymerase called DNA polymerase kappa (pol kappa), a member of the recently discovered Y family of DNA polymerases. The mouse PolK gene is expressed at high levels in the seminiferous tubules of the testis and in the adrenal cortex, and at lower levels in most other cells of the body including B lymphocytes. In vitro studies showed that pol kappa can act as an error-prone polymerase, although they failed to ascribe a clear function to this enzyme. The ability of pol kappa to generate mutations when extending primers on undamaged DNA templates identifies this enzyme as a potential candidate for the introduction of nucleotide changes in the immunoglobulin (Ig) genes during the process of SH. Here we show that pol kappa-deficient mice are viable, fertile and able to mount a normal immune response to the antigen (4-hydroxy-3-nitrophenyl)acetyl-chicken gamma-globulin (NP-GC). They also mutate their Ig genes normally. However, pol kappa-deficient embryonic fibroblasts are abnormally sensitive to killing following exposure to ultraviolet (UV) radiation, suggesting a role of pol kappa in translesion DNA synthesis.

Delayed anti-kappa response in kappa-deficient mice after neonatal, oral immunization with kappa-containing IgG

Antibody responses to kappa (κ)-light (L) chain are absent in normal (Cκ+/+) animals because of tolerance due to the abundance of κ-L chains expressed on more than 95% of all B cells and serum Ig. When heterozygous κ-sufficient (Cκ+/−) females are bred with homozygous κ-deficient (Cκ−/−) males, half of their offspring will become κ-deficient but have received κ-L chain containing maternal Ig, mainly IgG and IgA, through placental and intestinal transmission. The κ-containing maternal Ig persists for more than 2 months in the circulation of the offspring. Starting from weeks 15 to 20 of age, a spontaneous antibody response towards the maternal κ-L chains can be recorded. The time of onset, as well as the magnitude of the responses differ among individuals of the same litter. Invariably, once a response has been initiated, it transits into an IgG-type of response, which upon injection with κ-containing protein shows the features of a secondary type of immune response.

The level of expression of mu heavy chain modifies the composition of peripheral B cell subpopulations.

The B cell receptor (BCR) has a decisive role in transducing signals required for the development of B cells and their survival in the periphery. However, the processes that initiate these signals remain unclear and concepts of constitutive and ligand-dependent signaling have been proposed. Using a mu-transgenic mouse model, we have analyzed the impact of high surface IgM expression on the composition of the splenic B cell population. kappa-deficient mice homozygous for the H3-mu transgene have B cells with a higher BCR surface density than H3 heterozygous mice. This higher BCR expression is associated with an increase in the percentage and the total number of splenic B cells. In addition, an important proportion of CD23(-)CD21(+) marginal zone (MZ) B cells can be observed in H3 homozygous mice. However, these modifications operate in the absence of impairment of the positive selection process of the H3-mu/lambda1 combination over the H3-mu/lambda2 + 3 ones. These results suggest that (i) a constitutive BCR signaling directly correlated with BCR surface density is responsible for the efficient B cell colonization of the periphery with an accumulation of B cells in the MZ and (ii) a ligand-dependent BCR signal is responsible for the clonotype composition of the mature B cell repertoire.

The primary antibody repertoire of kappa-deficient mice is characterized by non-stochastic Vlamda1 + V(H) gene family pairings and a higher degree of self-reactivity.

We have investigated the primary antibody repertoire of genetically manipulated 129/Sv kappa-deficient (JCkappaD) mice, in order to understand the contributions of the lambda-light chain, in the absence of an otherwise predominant kappa-light chain, to the development of humoral immunity. The expression of Vlambda1 gene (lambda1 and lambda3 subtypes) and the Vlambda1 + V(H) (J558, 36-60, V(H)11 and S107) gene family associations were studied in 7.43 x 10(3) mitogen-activated splenic B-lymphocyte clones of JCkappaD origin. Furthermore, the functional significance of the exclusive expression of the lambda-light chain, in the peripheral B-cell repertoire of JCkappaD mice, was analysed by determining natural autoantibody specificities in the circulating serum immunoglobulin and the frequency of autoreactive B-lymphocyte clones in the peripheral B-lymphocyte repertoire. These experiments revealed that: first, of the three available Vlambda genes at the lambda locus, the Vlambda1 gene is the one that is expressed most frequently (59.9%); second, non-random Vlambda1 + V(H) (J558, 36-60) gene family pairings occur in kappa-deficient mice; and third, a higher degree of self-reactivity is generated as a result of exclusive use of the lambda-light chain, as evidenced by higher levels of serum natural autoantibodies as well as a high frequency of autoreactive B-lymphocyte clones in kappa-deficient (129/Sv JCkappaD) mice. These observations suggest that the high murine kappa/lambda ratio in mice may, apart from high sequence diversity at the kappa-locus, be a result of endogenous selection against the lambda-light chain to restrict self-reactivity within the homeostatic threshold.

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.

Contribution of the V lambda light chain to the development of the primary antibody repertoire.

The experiments outlined here provide evidence that most VH gene families are expressed in association with either of three V lambda genes available in kappa-deficient mice, though their expression is nonrandom. An exclusive use of V lambda L-chain genes, where predominantly expressed V lambda 1 and V lambda 2 differ by only seven amino acid substitutions in the germline, results in a higher degree of self-reactive antibodies to conserved natural antigens like albumin, thyroglobulin, and stress proteins. The higher levels of self-reactive antibodies, characteristic of preimmune B-cell repertoire, are likely to be a product of nonrandom VH gene family expression and their restricted pairing with three V lambda L-chain genes. These data suggest that a low lambda L-chain expression in normal mice is due, in part, to endogenous selection against lambda L chain in order to restrict self reactivity within the homeostatic threshold.

Antibody response against poly (Glu60Ala30Tyr10) terpolymer and bacterial levan in kappa-deficient mice.

In murine species, the kappa (+)-bearing immunoglobulins dominate the antibody (Ab) repertoire with a kappa/lambda ratio of 95:5. The aim of the present study is to investigate the characteristics of the antibody response in kappa-deficient (K-/-) mice immunized with a T-dependent synthetic antigen, poly(Glu60Ala30Tyr10) (GAT) and a T-independent antigen, bacterial levan (BL). K-/- mice were obtained by targeted deletion of the J kappa C kappa gene segments. In response to GAT, K-/- mice respond by producing increasing amounts of anti-GAT Ig lambda 1 and Ig lambda 2 in the primary as well as secondary response, although anti-GAT specific monoclonal antibodies (mAb) raised in K-/- mice are mostly of IgM isotype. The GAT public idiotype, GATIdX, present on all GAT-specific Ab bearing kappa light chain, is not detected in the sera of K-/- mice or on any of the anti-GAT lambda 1 mAb. In response to BL, the amount of Ig lambda 1+ Ab in K-/- mice is comparable to the amount of Ig kappa + Ab in normal mice. However, lambda 2+ Ab are detected neither in wild-type nor in K-/- mice. Like kappa + Ab, the majority of lambda 1+ mAb are specific for beta 2-6 fructosan present in BL and rye levan and, to some extent, express the BL-specific idiotype, A48ld. Our results show that important compensatory mechanisms occur in kappa-deficient mice, restoring their ability to mount immune responses against a variety of T-dependent and T-independent antigens by the alternative usage of the clonally restricted lambda repertoire.

Study of Self Reactive Antibodies in Kappa-Light Chain Deficient Mice

The kappa chain deficient mouse strain represents an excellent experimental system for studying the contribution of lambda light chains to the antibody repertoire. Here, we have studied the contribution of lambda chains to the generation of self reactive antibodies including RFs in kappa deficient mice with 129/sv background. These mutant mice produce rheumatoid factors similar to 129/sv mice and these antibodies are primarily encoded by lambda 2. This may be due to the production of RF by peritoneal B lymphocytes which belong to Ly1 B subset. Peritoneal B cell selectively produce lambda 2 and lambda 3 isotypes. Though lambda 1 positive RF is not detectable in the sera, lambda 1 positive specific precursor B cells are present in these mice and they can be activated by T-independent antigens. Our studies show that these mice also spontaneously produce anti-dsDNA antibodies bearing lambda 1 light chain but do not produce self reactive antibodies specific for eight different autoantigens. However, B cell precursors expressing lambda chains specific for autoantigens like collagen II, III, IV and histone 2A are present in the B cell repertoire of kappa-deficient mice. Thus, our results demonstrate that lambda light chain can compensate, to some extent, the lack of kappa chain repertoire, not only against foreign antigens, as observed previously, but also against a number of autoantigens.

Antibody response elicited by T-dependent and T-independent antigens in gene targeted kappa-deficient mice.

Animal models substantially contribute to the understanding of the pathogenesis of various human diseases, including those associated with genetic defects. Our study investigated the characteristics of antibody responses elicited by T-dependent and T-independent antigens in mice rendered kappa-deficient by targeted deletion of the J kappa C kappa gene segments. It is known that in normal murine species the kappa repertoire dominates the antibody repertoire (kappa/lambda ratio = 95:5). Our results indicate that the kappa gene deletion causes the alternative usage of lambda 1 (93%) and lambda 2 (7%) light chains, confirming previous studies demonstrating that in kappa-deficient mice all B cells express Ig lambda receptors. The anti-trinitrophenylbenzene (TNP) response in K-/- mice was compensated for by lambda 1 and lambda 2 bearing Igs. However, isoelectric focusing analysis of anti-TNP antibodies showed a considerably more restricted pattern of lambda anti-TNP antibodies in K-/- as compared with kappa antibodies in normal mice. No major differences were observed in the affinity for the hapten of kappa or lambda 1 or lambda 2 mAbs obtained from 129/Sv and K-/- mice. Furthermore, lambda 1 and lambda 2 chains can reconstitute the expression of an idiotype (460Id) borne on kappa anti-TNP antibodies. The 460Id was detected both in polyclonal and monoclonal anti-TNP antibodies obtained from K-/- mice. Our results clearly showed that the kappa anti-TNP repertoire is compensated by the lambda repertoire even though the latter is clonally restricted in K-/- mice.