The Role of IgM and Complement in Antibody Responses

An intact complement system including the complement receptors 1 and 2 (CR1/2) is crucial for the generation of a normal antibody response in animals and humans. Moreover, activation of the classical pathway is thought to be important since deficiency in complement components C1q, C2, C4 or C3 lead to impaired antibody responses. The classical pathway is mainly initiated by antibodies bound to their antigen. It is unclear how classical pathway activation can be crucial for primary antibody responses since the levels of specific antibodies are very low in naïve animals. It has been hypothesized that natural IgM, with high enough affinity, can initiate the classical pathway after immunization. To test this, we generated the knock-in mouse strain Cμ13, producing IgM unable to activate complement. Surprisingly, the antibody response against SRBC and KLH in Cµ13 mice was normal. Thus, the importance of classical pathway activation and natural IgM in antibody responses is not dependent on the ability of IgM to activate complement. SIGN-R1, SAP and CRP are other known activators of the classical pathway, but mice lacking these also had normal antibody responses. Complement activation leads to the generation of C3 split products which are ligands for CR1/2. In mice, CR1/2 are expressed on B cells and follicular dendritic cells (FDC), but it is unclear on which cell-type expression of CR1/2 is needed for the generation of a normal antibody response. Some reports argue that increased antigen retention by CR1/2+ FDC would increase the effective antigen concentration, giving more effective B-cell stimulation. In contrast, several mechanisms involving CR1/2 on B cells are suggested. First, marginal zone B cells could transport complement-coated antigen or IC via CR1/2 into the follicle. Second, different ways of co-crosslinking the B-cell receptor with CR1/2, lowering the threshold for B-cell activation, have been proposed. Finally, CR1/2 on B cells are shown in vitro to facilitate endocytosis and thereby presentation of antigen to T cells. We show that abrogated antibody responses in mice lacking CR1/2 are not due to lack of CR1/2-mediated antigen presentation to T cells. Chimeric mice with CR1/2 expression on both B cells and FDC, on neither B cells nor FDC, or on either B cells or FDC, were generated. The antibody response against SRBC was completely dependent of CR1/2-expression on FDC. However, when this requirement was fulfilled, B cells without expression of CR1/2 were equally efficient antibody producers as wildtype B cells. Antigen-specific IgM together with its antigen can enhance the antibody response to that antigen and CR1/2-expression is crucial for the enhancement. We show that the response to IgM in complex with SRBC is dependent on CR1/2 expression on both B cells and FDC

Complement-Activating IgM Enhances the Humoral but Not the T Cell Immune Response in Mice

IgM antibodies specific for a certain antigen can enhance antibody responses when administered together with this antigen, a process believed to require complement activation by IgM. However, recent data show that a knock-in mouse strain, C mu 13, which only produces IgM unable to activate complement, has normal antibody responses. Moreover, the recently discovered murine IgM Fc receptor (Fc mu R or TOSO/FAIM3) was shown to affect antibody responses. This prompted the re-investigation of whether complement activation by specific IgM is indeed required for enhancement of antibody responses and whether the mutation in C mu 13 IgM also caused impaired binding to Fc mu R. The results show that IgM from C mu 13 and wildtype mice bound equally well to the murine Fc mu R. In spite of this, specific C mu 13 IgM administered together with sheep red blood cells or keyhole limpet hemocyanine was a very poor enhancer of the antibody and germinal center responses as compared with wildtype IgM. Within seconds after immunization, wildtype IgM induced deposition of C3 on sheep red blood cells in the blood. IgM which efficiently enhanced the T-dependent humoral immune response had no effect on activation of specific CD4+ T cells as measured by cell numbers, cell division, blast transformation, or expression of the activation markers LFA-1 and CD44 in vivo. These observations confirm the importance of complement for the ability of specific IgM to enhance antibody responses and suggest that there is a divergence between the regulation of T-and B-cell responses by IgM

High-Resolution Genotyping of Chlamydia trachomatis Strains by Multilocus Sequence Analysis

Genotyping of Chlamydia trachomatis is limited by the low sequence variation in the genome, and no adequate method is available for analysis of the spread of chlamydial infections in the community. We have developed a multilocus sequence typing (MLST) system based on five target regions and compared it with analysis of ompA, the single gene most extensively used for genotyping. Sequence determination of 16 reference strains, comprising all major serotypes, serotypes A to L3, showed that the number of genetic variants in the five separate target regions ranged from 8 to 16. The genetic variation in 47 clinical C. trachomatis isolates of representative serotypes (14 serotype D, 12 serotype E, 11 serotype G, and 10 serotype K strains) was analyzed; and the MLST system detected 32 variants, whereas 12 variants were detected by using ompA analysis. Specimens of the predominant serotype, serotype E, were differentiated into seven genotypes by MLST but into only two by ompA analysis. The MLST system was applied to C. trachomatis specimens from a population of men who have sex with men and was able to differentiate 10 specimens of one predominant ompA genotype G variant into four distinct MLST variants. To conclude, our MLST system can be used to discriminate C. trachomatis strains and can be applied to high-resolution molecular epidemiology

01_A_S3A_069

Κωδικός τεκμηρίου: 01_A_S3A_069-019. Είδος τεκμηρίου: SLIDES 10Χ12,5 Χ 1 ΚΑΡΕ - ΕΓΧΡΩΜΑ. Ανήκει σε: 01_A_S3A_069 - ΚΟΥΤΙ "ΔΙΑΦΑΝΕΙΕΣ ΜΕΓ.

CR1/2 on FDCs are required for a robust IgG anti-SRBC response to SRBC.

<p>BALB/c and <i>Cr2^−/−</i> mice were irradiated and reconstituted with either BALB/c or <i>Cr2^−/−</i> bone marrow. Six weeks after reconstitution, mice (n = 6/group) were immunized i.v. with 5×10⁶, 5×10⁷, or 5×10⁸ SRBC. All mice were bled at indicated time points. Sera were diluted 1∶125 (A) or 1∶625 (B and C) and screened for IgG anti-SRBC in ELISA. P-values represent comparisons between the responses in recipients with the same background; ns = p>0.05; * = p<0.05; ** = p<0.01; *** = p<0.001. Representative of two (A) or one (B, C) experiments.</p

CR1/2 on B cells and FDCs is required for optimal antibody responses to IgM-SRBC complexes.

<p>BALB/c and <i>Cr2^−/−</i> mice were irradiated and reconstituted with either BALB/c or <i>Cr2^−/−</i> bone marrow. Six weeks after transplantation, mice were immunized with 5×10⁵ (A–D) or 5×10⁶ (E–H) SRBC alone (open squares) or together with IgM anti-SRBC with a hemagglutination titer of 1∶32 (filled squares) or with IgM anti-SRBC alone (open triangles) (n = 6/group). All mice were bled at indicated time points. Sera were diluted 1∶25 (A–D) or 1∶625 (E–H) and screened for IgG anti-SRBC. Two statistical comparisons were made, both using Student's <i>t</i>-test. First, comparisons between the responses in mice immunized with SRBC alone versus IgM and SRBC (to determine whether IgM enhanced antibody responses significantly; filled versus open symbols), where ns = p>0.05; * = p<0.05; ** = p<0.01; *** = p<0.001. Second, comparisons between the responses between various chimeras immunized with IgM-SRBC (to determine whether CR1/2⁺ B cells contributed significantly to the antibody response to IgM-SRBC in mice with CR1/2⁺ FDCs (A vs B; E vs F) and CR1/2⁻ FDCs (C vs D; G vs H)), where ns = p>0.05; ° = p<0.05; °° = p<0.01; °°° = p<0.001. For graphic clarity, non-significant differences are not indicated. Representative of one (A–D) and two (E–H) experiments.</p

IgM from BALB/c mice enhances germinal center responses.

<p>On day 0, BALB/c mice were immunized i.v. with WT or Cµ13 IgM specific for KLH (50 µg/mouse) or SRBC (0.2 ml of a solution with HA titer 1:32) 30 min before 10 µg KLH (A), 5×10⁵ (B) or 5×10⁶ SRBC (C) were administered via the same route; controls received antigens or specific IgM alone. Spleens were harvested on day 10. Splenocytes from half of each spleen were analyzed by flow cytometry; germinal center B cells were gated as GL7⁺PNA⁺ amongst B220⁺ cells (Figure S1) and the percentages of germinal center B cells were quantified (A-C, upper left panels). The other halves of the spleens were sectioned, stained with anti-B220 (blue), anti-MOMA (green) and PNA (red), and analyzed for number of PNA⁺ germinal centers in B cell follicles by confocal microscopy (A-C, upper right panels); each image is a representative area (1725 µm × 1295 µm) for 2-3 whole sections with original magnification ×10 (A-C, lower panels). Germinal center responses of mice immunized with specific IgM alone were always lower than the responses of mice immunized with antigens alone (not shown). Data are representative of two experiments with each antigen dose. ns = not significant; * = p < 0.05; ** = p < 0.01.</p

WT IgM induces rapid deposition of C3 on SRBC in the blood.

<p>BALB/c mice were immunized i.v. with IgM anti-SRBC (0.2 ml of a solution with HA titer 1:32) from WT BALB/c (WT IgM; n = 2) or Cµ13 mice (Cµ13 IgM; n = 2). Thirty minutes later, 5×10⁸ SRBC were administered. Mice given 5×10⁸ SRBC alone were used as controls (n=2). Peripheral blood samples were taken 1 min after immunization and immediately mixed with 1 µl lepirudin (50 mg/ml). Rabbit IgG anti-SRBC was used to identify SRBC in the blood (left panel). The deposition of C3 on SRBC in the blood from mice immunized with SRBC alone (black) or together with WT IgM (red) or Cµ13 IgM (blue) was analyzed (right panel). Representative of three independent experiments.</p

Activation of antigen-specific CD4⁺ T cells is not enhanced by specific IgM.

<p>BALB/c mice were adoptively transferred with 15×10⁶ splenocytes from DO11.10 mice. The next day, mice were immunized with 5×10⁶ SRBC-OVA alone or together with WT IgM anti-SRBC (0.2 ml of a solution with HA titer 1:8), 5×10⁸ SRBC-OVA (positive controls) or 5×10⁸ unconjugated SRBC (negative controls). One, 2, 3, and 4 days after immunization, spleens from 2-3 mice per group were harvested and analyzed by flow cytometry. Kinetics of KJ1-26⁺CD4⁺ cell expansion (A), percentages of blasts among KJ1-26⁺CD4⁺ cells (B, left panel) and expression of surface markers LFA-1 (C, left panel) and CD44 (D, left panel) on KJ1-26⁺CD4⁺ cells were quantified. Numbers in histograms indicate the percentages of blasts, LFA-1^high and CD44^high cells among KJ1-26⁺CD4⁺ cells in the positive controls. Representative histograms (B-D, right panels) of each group immunized with 5×10⁸ SRBC-OVA (black), 5×10⁸ unconjugated-SRBC (grey), 5×10⁶ SRBC-OVA alone (blue) or together with WT IgM anti-SRBC (red) are shown for day 3. SEM were frequently too small to be seen in the figures.</p