CCR7-deficient mice develop atypically persistent germinal centers in response to thymus-independent type 2 antigens.

Thymus-independent type 2 (TI-2) antigens are repetitive antigens capable of eliciting antibody responses without T cell help. They are important in the immune response against encapsulated bacteria and as a rapid first line of defense against pathogens. TI-2 antigens induce strong proliferation in extrafollicular foci. However, any germinal centers forming in response to TI-2 antigens involute synchronously 5 days after immunization. This is thought to be caused by the lack of T cell help. Surprisingly, immunization of mice deficient for the homeostatic chemokine receptor CCR7 with TI-2 antigens resulted not only in the expected, vigorous extrafollicular plasma cell response but also in persisting splenic germinal centers. This was observed for two different TI-2 antigens, heat-killed Streptococcus pneumoniae and (4-hydroxy-3-nitrophenyl)acetyl-Ficoll (NP-Ficoll). Germinal centers induced by TI-2 and thymus-dependent (TD) antigens were located in the periarteriolar area of the white pulp in CCR7 knockout mice, corresponding to the T zone of wild-type (WT) mice. The TI-2-induced germinal centers contained peripheral rings of follicular dendritic cells and unusually for TI-2-induced germinal centers, T cells. The licensing responsible for their atypical persistence did not endow TI-2-induced germinal centers with the full range of characteristics of classic germinal centers induced by TD antigens. Thus, class-switching, affinity maturation, and memory B cell generation were not increased in CCR7-deficient mice. It seems unlikely that a defect in regulatory T cell (Treg) location was responsible for the atypical persistence of TI-2-induced germinal centers, as Tregs were comparably distributed in germinal centers of CCR7-deficient and WT mice.

Antibody structural variation in rainbow trout fluids.

Rainbow trout (Oncorhynchus mykiss) were immunized with trinitrophenylated-keyhole limpet hemocyanin (TNP-KLH) and the redox structure of induced anti-TNP antibodies from the serum, mucus, egg and ovarian fluid was examined. In conducting these studies it was determined that all TNP-specific antibody from each source possessed the mAb-specific H chain (1-14) epitopes, which facilitated the direct structural analysis of the induced antibodies. A protocol was developed which ensured complete adsorption of all specific anti-TNP antibody from each fluid. Together these protocols permitted the unbiased compositional analysis of all redox forms of the anti-TNP antibodies from each source. All antibodies, regardless of source, possessed the same molecular mass, characteristic of the trout tetramer (800 kDa). It was found that specific antibody titers were significantly higher in male than female trout, while the degree of disulfide polymerization was relatively invariant in male antibodies, while being highly variable in female antibodies. Within the females, no distinctively different redox ratios were between antibodies isolated from sera, ovarian fluid or eggs: however, mucus antibodies possessed a unique redox structure consisting of halfmeric constituents that were not observed in antibodies from other fluids.

The Rac2 guanosine triphosphatase regulates B lymphocyte antigen receptor responses and chemotaxis and is required for establishment of B-1a and marginal zone B lymphocytes.

We have defined roles for the hemopoietic-specific Rho guanosine triphosphatase, Rac2, in B lymphocyte development and function through examination of rac2(-/-) mice. Rac2-deficient mice displayed peripheral blood B lymphocytosis and marked reductions in peritoneal cavity B-1a lymphocytes, marginal zone B lymphocytes, and IgM-secreting plasma cells as well as reduced concentrations of serum IgM and IgA. The rac2(-/-) B lymphocytes exhibited reduced calcium flux following coligation of B cell AgR and CD19 and reduced chemotaxis in chemokine gradients. T cell-independent responses to DNP-dextran were of reduced magnitude, but normal kinetics, in rac2(-/-) mice, while T-dependent responses to nitrophenyl-keyhole limpet hemocyanin were subtly abnormal. Rac2 is therefore an essential element in regulating B lymphocyte functions and maintaining B lymphocyte populations in vivo.

Deficiency in Msh2 affects the efficiency and local sequence specificity of immunoglobulin class-switch recombination: parallels with somatic hypermutation.

During maturation of the immune response, IgM+ B cells switch to expression of one of the downstream isotypes (IgG, A or E). This class switching occurs by region-specific recombination within the IgH locus through an unknown mechanism. A lack of switch recombination in mice deficient in components of the DNA-dependent protein kinase (DNA-PK)-Ku complex has pointed to a role for non-homologous end joining. Here we characterize a switching defect in mice lacking a protein involved in DNA mismatch recognition. Mice deficient in Msh2 give diminished IgG (but not IgM) responses following challenge with both T cell-dependent and T cell-independent antigens. This appears to reflect a B cell-intrinsic defect since B cells from Msh2-deficient mice also exhibit impaired switching (but not blasting or proliferation) on in vitro culture with lipopolysaccharide. Furthermore, those switches that do occur in Msh2-deficient B cells reveal a shift in the distribution of recombination sites used: the breakpoints are more likely to occur in consensus motifs. These results, which intriguingly parallel the effects of Msh2 deficiency on hypermutation, suggest a role for Msh2 in the mechanics of class-switch recombination.

Dysregulated intrathymic development in the IL-2-deficient mouse leads to colitis-inducing thymocytes.

Gene-targeted mice lacking the IL-2 gene (IL-2 -/- mice) develop various forms of autoimmunity as well as severe colitis, either spontaneously in a conventional environment or after immunization with 2,4,6-trinitrophenol (TNP)-conjugated keyhole limpet hemocyanin (KLH) in a specific pathogen-free environment. We show here that the induction of colitis with TNP-KLH induces a change in the thymocyte population characterized by decreased numbers of double positive (DP; CD4+CD8+) thymocytes (IL-2 +/+, 45.2 x 10(6) vs IL-2 -/-, 23.6 x 10(6)) and increased numbers of single positive (SP; CD4+CD8- or CD4-CD8+) thymocytes (IL-2 +/+, 5.3 x 10(6) vs IL-2 -/-, 20.9 x 10(6)). The latter also bear activation markers. In addition, thymocytes from TNP-KLH-immunized IL-2 -/- mice produce more IFN-gamma and less IL-4 than similarly immunized IL-2 +/+ mice. These defects in thymocyte maturation and lymphokine production are IL-12 driven, since they are prevented when immunized IL-2 -/- mice are coadministered with anti-lL-12. Furthermore, we demonstrate that IL-2 -/- mice exhibit decreased cortical apoptosis as determined by thymocyte numbers and detection of apoptotic cells in situ. Finally, we show that colitis-inducing thymocytes are generated in the immunized IL-2 -/- thymus, since IL-2 +/+ mice develop colitis following injection of small numbers of single positive thymocytes from immunized IL-2 -/- mice but not from IL-2 +/+ mice. Taken together, these data indicate that, in the absence of IL-2, thymocyte maturation is abnormally directed by IL-12 toward the generation of mature, activated Th1-type thymocytes that are capable of mediating colitis.

Carrier-independent hapten recognition and promiscuous MHC restriction by CD4 T cells induced by trinitrophenylated peptides.

The elucidation of mechanisms underlying the recognition of haptens by class II MHC-restricted T cells is instrumental for the understanding of chemical- and drug-induced allergies. We have previously demonstrated that trinitrophenyl (TNP) peptides represent dominant antigenic epitopes for CD8+ and CD4+ mouse T cells triggered by chemically TNP-modified APC. Here, we report the characterization of TNP-specific, CD4+ mouse T cell lines and hybridomas that were induced in vivo and in vitro by defined hapten-conjugated peptides. These peptides, which we had previously shown to induce contact sensitivity to picryl chloride in vivo regardless of sequence homologies to mouse proteins, were found to activate carrier-independent TNP-specific T cells in vitro. We interpret these findings to support our view that carrier-independent T cells, reactive to particularly repetitive hapten epitopes, may play a crucial role in allergies to chemicals and drugs. In addition to carrier independence, one of our hybridomas (IT-H6/A11) exhibited a striking promiscuity of MHC restriction. Although absolutely dependent in its TNP reactivity on the presence of MHC class II molecules, the IT H6/A11 hybridoma completely ignored class II polymorphism and even reacted to TNP peptides presented on human DR molecules. Regarding hapten allergies in humans with a heterozygous situation for three types of class II molecules (DR, DP, and DQ), such promiscuous MHC restriction should lead to the presentation of even higher epitope densities to the respective T cell clones. Hybridoma IT-H6/A11, reacting to TNP independent of carrier peptide and of MHC haplotype, also allowed for an unusually systematic study of the minimal requirements for TNP recognition. Despite an almost complete ignorance of amino acid side chains on the carrier peptide, our data indicate a clearly position-specific interaction of hapten and TCR.

bcl-2 alters the antigen-driven selection of B cells in mukappa but not in mu-only Xid transgenic mice.

A point mutation in the pleckstrin homology domain of the mouse Bruton's tyrosine kinase (btk) gene results in an X-linked immune defect, Xid, characterized by immunologic unresponsiveness to polymeric carbohydrate Ags. In Xid mice, B cells specific for phosphocholine (PC) do not develop in peripheral lymphoid tissues because they either fail to be positively selected from the marrow or they are clonally deleted via an Ag-driven, receptor-mediated process. Overexpression of the bcl-2 gene allows PC-specific B cells to survive and mature in Xid mukappa anti-PC transgenic mice, but PC-specific B cells are not rescued by bcl-2 in Xid mu-only transgenic mice. The failure of bcl-2 to rescue PC-specific B cells, in mu-only transgenic mice suggests that either it does not correct the btk defect in the Ag-driven selection process that occurs in pre-B cells and/or in very immature B cells or that a btk-dependent proliferative phase is required for the selection and amplification of the PC-specific B cells in mu-only transgenic mice. The rescue of PC-specific B cells in mukappa transgenic mice indicates that bcl-2 can alter receptor-mediated B cell selection at late stages in B cell development. The rescued PC-specific B cells in Xid male mice do not exhibit an altered proliferation profile in response to B cell-stimulating agents compared with B cells from unmanipulated Xid mice; thus, they fail to respond to soluble anti-mu, or PC-dextran, but they proliferate in response to PC, anti-mu, or anti-id conjugated to Sepharose.

Stimulation by T cell independent antigens can relieve the arrest of differentiation of immature auto-reactive B cells in the bone marrow.

The pair of microH-chain and kappa L-chain transgenes encoding the Sp6 TNP/DNA-specific IgM was bred onto the rearrangement-deficient genetic background of RAG-2T mice, and onto the kappa L-chain expression-deficient background of iE kappa T mice. Bone marrow of Sp6 transgenic RAG-2T mice contained normal numbers of B220(CD45R)+c-kit+ pro/preB-I-like cells and normal numbers of B220(CD45R)+TAC+ preB-II-like cells. Most strikingly, the numbers of immature sIgM+ B cells in the bone marrow were at least five-fold lower than normal, while mature B cells were almost undetectable in bone marrow as well as spleen. Hence, B cell development in these mice appears to be arrested at the transition from preB-II to immature B cells. The contents of bone marrow and spleen of the different precursors, immature and mature B cell compartments in Sp6iE kappa T mice were found to be similar to those of normal mice except that all sIg+ cells expressed lambda L-chains, of which 40% coexpressed the transgenic kappa L-chain. It indicates that the repertoire of lambda L-chain rearrangements and the lambda L-chains expressed from it suffices to relieve the arrest of differentiation seen in Sp6RAG-2T mice. The T cell-independent antigen TNP-Ficoll elicited within 5 days a response of the Sp6RAG-2T mice to develop to IgM-secreting cells and to fill the serum pool with the Sp6 transgenic IgM to 100 micrograms/ml, i.e. to normal serum levels of IgM in normal mice. TNP-Ficoll appears to interfere with the arrest of differentiation. Two scenarios for this arrest of differentiation and its relief by the T-independent antigen TNP-Ficoll are discussed.

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.

Strain differences of age-dependent changes in the responsiveness to a T-independent type-2 antigen in mice.

We have assessed age-associated early changes in antibody response to a T-independent type-2 (TI-2) antigen, dinitrophenylated ficoll (DNP-Ficoll). Mice of most strains, including long-lived and autoimmune-prone strains, give a high response when approximately 2 months old; thereafter the response declines sharply to the 3rd-4th month of age and continues to do so, more gradually, up to the age of 6 months. Age-related changes in the response of C57BL/6 mice follows a different course: the response remains unchanged up to the first year of life, i.e. to middle age. The in vitro anti-DNP-Ficoll antibody response of B cells could be increased by the addition of young syngeneic T cells. The augmenting activity of splenic T cells of C3H/He mice declines clearly as a function of age. In contrast, splenic T cells of C57BL/6 mice have low augmenting activity whether the T cells are obtained from young or middle-aged donors. Unlike the augmenting capacity of T cells, B-cell responsiveness to DNP-Ficoll increases until middle age in all strains examined. We conclude that early age-associated changes in antibody response to the TI-2 antigen is polymorphic and that the early age-related decline in in vivo responsiveness is attributable to an age-associated decline in augmenting T helper cell activity.

Mechanism of immune dysfunction associated with minor antigen graft-vs-host disease in mice.

A well-characterized murine model of graft-vs-host disease (GVHD) that develops in response to minor histocompatibility antigens was used to study the mechanism of an immunodeficiency syndrome that is associated with GVHD. Lethally irradiated mice were transplanted with a combination of bone marrow and spleen cells from H-2 compatible donors that differed at multiple minor histocompatibility antigens, or from syngeneic donors. Four to 12 weeks later, the humoral responses of transplanted and control mice to the T dependent antigens bacteriophage phiX174 and TNP-sheep red blood cells (TNP-SRBC), and to the T independent antigen TNP-Brucella abortus (TNP-BA) were determined. The results demonstrate that mice with GVHD have relatively intact B Cell function and a profound defect in T helper cell function. The immune response to T dependent antigens normalized with repeated immunization. We conclude that immune dysfunction in mice with GVHD is due to a reversible defect in T helper cell function.