Quinoline-3-carboxamides modulate primary T cell-dependent B cell responses but do not inhibit functional immunity.

The effect of a quinoline-3-carboxamide on the T cell-dependent B cell response was investigated in C57BL/6 mice after NP-CGG immunization. The primary serum response to the hapten was slightly inhibited by treatment with a quinoline-3-carboxamide. This inhibition was paralleled by reduced numbers of germinal centre (GC) B cells and follicular T cells in the spleen up to 21 days after immunization. Also, both the number of GCs formed and their size were reduced by quinoline-3-carboxamide treatment. In contrast to the observation in the primary immune response, there was no inhibitory effect on the secondary immune response. These data could help to explain how quinoline-3-carboxamides can modulate immune function in autoimmune diseases without being immunosuppressive.

Analysis of antigen-specific and naturally occurring IgM and IgA steady-state levels in J-chain negative C57BL/6 mice.

Naturally occurring IgM and IgA levels are remarkably stable between different individuals. In mice lacking joining chain (J-chain), the steady-state levels of IgM are reduced, while IgA levels are elevated. We have here analysed the IgM and IgA responses as well as the regulation of naturally occurring antibodies in mice that delete all J-chain expressing B cells (JDTA mice) and have been back-crossed to C57BL/6 mice. The IgM response to a T-cell-dependent antigen was reduced in JDTA mice but still easily detectable. In contrast, a very pronounced primary IgA response could be detected in JDTA mice while wild type controls showed no detectable primary IgA response. With regard to naturally occurring antibodies, bone marrow chimeras between JDTA and control C57BL/6 mice had a donor cell phenotype with regard to serum IgM and IgA. Mixed bone marrow chimeras had an intermediate phenotype, indicating that the naturally occurring antibody IgM and IgA levels are B-cell autonomous and not subjected to feed-back control. This was confirmed by transfer of the dominant naturally occurring IgM/IgA phenotype to the recipient by peritoneal exudate cells.

T-cell receptor repertoire in human germinal centres.

A search for an antigen-driven expansion of T lymphocytes in the inflamed joints in rheumatoid arthritis (RA) patients have been going on for decades. We here analyzed the human germinal centre T-cell receptor (TCR) Vbeta gene usage with polymerase chain reaction (PCR) combined with sequence analysis, to address the question of clonality in tonsils and synovial tissue from RA patients. Our data show a large degree of TCR heterogeneity in both these histological structures. Furthermore, clonally related T cells were found within different closely located germinal centres indicating either an active T-cell migration between germinal centres (GC) or that a T-cell clone may seed more than one GC.

Joining chain-expressing and -nonexpressing B cell populations in the mouse.

The diphtheria toxin A chain (DTA) was gene targeted into the Joining chain (J chain) locus to create a mouse strain selecting against J chain-expressing cells, JDTA mice. Serum immunoglobulin (Ig)M and serum IgG were reduced six to eightfold, while serum IgA was elevated 14-fold in these mice. JDTA mice were immune competent although the serum Ig response compared with wild-type mice was reduced sixfold at day 14 but only fourfold at day 45 after immunization. Exchanging the DTA gene with a cDNA for c-myc resulted in mice with a distinct phenotype with increased Ig production and enhanced humoral immune responses. Analysis of single B cells stimulated by lipopolysaccharide in vitro using reverse transcription-polymerase chain reaction showed that J chain-nonexpressing B cells could be detected that had a secretory phenotype as determined by an abundance of transcript for secretory IgM. Finally, limiting dilution analysis of peripheral B cells showed that J chain expression was a clonal property already established in naive, peripheral B lymphocytes.

Change in the Th1/Th2 phenotype of memory T-cell clones from rheumatoid arthritis synovium.

The stability of established memory T helper (Th)1/Th2 cells in chronic inflammatory diseases is not clear, and a shift of the cytokine balance could control chronic inflammation. In order to study the regulation of the Th phenotype of memory T cells, polyclonal T-cell lines and clones with a Th1, Th0 or Th2 phenotype were developed from rheumatoid synovial tissue. Th1 [interleukin (IL)-12 + anti-IL-4] and Th2 (IL-4 + anti-IL-12) promoting environments and IL-2 were used to manipulate the cytokine profile. Polyclonal T-cell lines of predominantly Th1 type could be shifted to produce Th2 cytokines, and polyclonal Th2/Th0 lines could be shifted to produce Th1 cytokines. However, this shift was due to an amplification of CD8+ T cells with a memory phenotype and a loss of the CD4+ T cells, giving Tc2 or Tc1 profiles, respectively. Th2 clones cultured repeatedly with IL-2 switched to either a Th0 or a Th1 phenotype, while both Th1 and Th0 memory clones kept a stable phenotype. Addition of Th2-promoting conditions strongly reduced the production of both interferon-gamma and IL-17, while Th1-promoting conditions increased the production of these cytokines. These results demonstrate that RA Th2 clones readily switch, while Th1 and Th0 clones are stable. However, induction of Th2 cytokines can be obtained in polyclonal polarized memory T cells due to amplification of Tc2 cells.

Somatic mutation of immunoglobulin V genes in vitro.

The molecular mechanism behind affinity maturation is the introduction of point mutations in immunoglobulin (Ig) V genes, followed by the selective proliferation of B cells expressing mutants with increased affinity for antigen. An in vitro culture system was developed in which somatic hypermutation of Ig V genes was sustained in primed B cells. Cognate T cell help and cross-linking of the surface Ig were required, whereas the addition of lipopolysaccharide or a CD40 ligand to drive proliferation was insufficient. This system should facilitate understanding of the molecular and cellular mechanisms that regulate somatic mutation and B cell selection.

The effect of carrier and carrier priming on the kinetics and pattern of somatic mutation in the V chi Ox1 gene.

Priming mice with a chicken gamma globulin (CGG) carrier protein significantly accelerated the onset of somatic mutation in the V chi Ox1 gene when the mice were subsequently immunized with 2-phenyl-5-oxazolone (phOx) coupled to CGG. The first mutations were already detected 7 days after immunization, while in the true primary response, they are not apparent until day 10. It was also found that comparing the mutation pattern of V chi Ox1 genes from hybridomas derived after immunization with phOx coupled to different carriers revealed quite distinct patterns of somatic mutation. Analysis of hybridoma sequences from the primary immune response to phOx-ovalbumin showed that the codons for Ser29, Ser31 and Lys45 were hot-spots for somatic mutation. Thus, the frequency and pattern of somatic mutations in the V chi Ox1 gene depends on the available T cell help as well as on the complex structure of the immunizing antigen.

Cell-cycle kinetics of proliferating mouse B lymphocytes in vitro.

Resting mouse B lymphocytes were stimulated in vitro with lipopolysaccharide, Sepharose-coupled anti-kappa antibodies or a combination of the two. B lymphocytes stimulated with anti-kappa entered the cell-cycle with more rapid kinetics and at a higher frequency than did the corresponding cell population stimulated with lipopolysaccharide. Using cell cycle analysis after DNA staining combined with an M phase block, the cell-cycle kinetics of in vitro cultured B-lymphocytes was studied. The labelling index of lipopolysaccharide stimulated B lymphocytes was 60% while that for anti-kappa Sepharose stimulated cells was 85%. The generation time of the actively cycling population from both types of cultures was constant and was of the order of 18 h. Thus, the fraction of B lymphocytes induced to proliferate in vitro varies depending on the stimulus, while the growth kinetics of the actively proliferating populations are remarkably constant.

Kinetics of somatic mutation in lymph node germinal centres.

Somatic mutation activity in immunoglobulin V kappa genes during the response to the hapten 2-phenyl-5-oxazolone was measured in lymph node B-cell populations at various timepoints after footpad immunization. When the V kappa Ox1 genes rearranged to the J kappa 5 segment were amplified from genomic DNA using the polymerase chain reaction and sequenced, somatic mutations could be detected as early as day 4 after immunization. Somatic mutations were also detected after sequencing RNA from oxazolone-specific hybridomas derived from lymph node cells at day 4 after immunization. These early mutations were found mostly in cells with a germinal centre phenotype. No indication of selection at the population level by apoptosis was detected until day 7 after immunization. These results suggest somatic mutations can be induced very early during the immune response in lymph node cells, prior to the peak of clonal expansion and selection with regard to antigen binding.

Analysis of somatic mutation activity in multiple V kappa genes involved in the response to 2-phenyl-5-oxazolone.

We have studied somatic mutation activity early in a response to 2-phenyl-5-oxazolone coupled to ovalbumin (phOx-OVA). Although the V kappa Ox1 gene rearranged to J kappa 5 is known to predominate in this response, other closely related V kappa genes are involved. We compared the introduction of point mutations into V kappa Ox1 genes and into a set of related V kappa genes rearranged to the same J kappa segment at two time points after primary immunization. The result showed that quantitation of mutations in a single rearrangement substrate leads to an underestimation of the total mutational activity. There is pronounced somatic mutation activity early within genes that may be absent later in the response. We also show that multiple somatic mutations can be detected in B cells from draining lymph nodes after foot-pad injection with phOx-OVA already at day 7 after immunization. The data suggest a system in which mutation acts early in the response on a wide range of substrates and that selection and expansion of high affinity paratopes occurs later.