Early B-cell and plasma cell populations in bone marrow of unimmunized poly(ADP-ribose) polymerase-1 (Parp-1)-deficient (Parp-1^−/−) mice. (a) Mononuclear cells derived from bone marrow were stained with anti-B220 and anti-immunogobulin M (IgM) and live-gated cells were analysed. The two B-cell populations B220^high and B220^low were similar in wild-type and Parp-1^−/− mice. Experiments were carried out in five animals and similar results were obtained. (b) In order to quantify plasma cells, bone marrow-derived mononuclear cells were stained with anti-B220, anti-IgM and anti-CD138 (syndecan-1). CD138 expression on B220⁻IgM⁻ cells (gate defined by dotted line in a) is presented. Plasma cells (B220⁻IgM⁻CD138⁺) in Parp-1^−/− mice were significantly reduced by about 70% compared with normal animals (P = 0.002; Mann–Whitney test). Experiments were carried out in five animals and similar results were obtained.

Basal antibody expression in vivo and antibody expression after culture of splenic B cells with growth factors. (a) Basal levels of serum immunoglobulin in a group of six poly(ADP-ribose) polymerase-1 (Parp-1)-deficient (Parp-1^−/−) mice and six wild-type litter mates were measured by enzyme-linked immunosorbent assay (ELISA). There were significant differences in the level of immunoglobulin G2a (IgG2a) (P = 0·009; Mann–Whitney U-test), IgG2b (P = 0·041; Mann–Whitney test) and IgA (P = 0·004; Mann–Whitney test). (b) Immunoglobulin levels produced after 5 days of in vitro culture of purified mouse splenic B cells stimulated with lipopolysaccharide (LPS), LPS and interleukin (IL)-4 (IgG1), LPS and interferon (IFN)-γ (IgG2b) and LPS and transforming growth factor (TGF)-β (IgA)30 were measured. There were significant differences in the levels of IgG2a (P = 0·041; Mann–Whitney test) and IgA (P = 0·009; Mann–Whitney test).

Activation-induced cytidine deaminase (AID) expression, proliferation and poly(ADP-ribose) polymerase (PARP) activity. (a) AID expression was measured by real-time polymerase chain reaction (PCR). Splenic B cells were cultured with lipopolysaccharide (LPS) and harvested after 3 and 7 days. Expression levels were normalized to day 3 levels in B cells from normal mice. Similar results were obtained from splenic B cells from two normal and two Parp-1-deficient animals. Black bars represent wild-type mice and open bars Parp-1^−/− animals. (b) B cells from Parp-1^−/− mice showed normal proliferative responses. B cells from six Parp-1-deficient and six wild-type mice were cultured in the absence or presence of LPS and proliferation measured as [³H]thymidine incorporation. Results represent the mean ± standard errors of the mean obtained from four independent experiments carried out in triplicate. (c) PARP activity was measured in purified B cells from Parp-1^−/− and Parp-2^−/− mice. Compared with wild-type animals, PARP activity was reduced by about 50% in Parp-1^−/− mice but was normal in Parp-2^−/− mice. c.p.m., counts per minute.

Lymphocyte populations in unimmunized 8–10-week-old poly(ADP-ribose) polymerase-1 (Parp-1)-deficient (Parp-1^−/−) mice. (a) Splenic B cells were stained with anti-B220 to identify B cells and anti-CD3 to identify T cells. The numbers of B cells were similar in wild-type (WT) and Parp-1^−/− mice, although there was a decrease in the proportion of B cells in knock-out animals as a result of a corresponding increase in T cells. (b) To assess T-cell subsets, splenic B cells were stained with anti-CD3, anti-CD4 and anti-CD8. Live-gated, CD3⁺ cells were analysed. Parp-1^−/− mice had normal numbers of CD4 and CD8 T cells. Experiments were carried out in four animals and similar were results obtained. (c) Comparison of total numbers of B and T cells. The percentages from (a) were multiplied by the total number of splenic B cells to give the absolute numbers of B and T cells. The mean ± standard error of the mean from analysis of four mice is presented. Parp-1^−/− mice had significantly increased numbers of T cells (P = 0·008; Mann–Whitney test) but B-cell numbers did not differ significantly from those in wild-type animals.

B-cell subsets in unimmunized poly(ADP-ribose) polymerase-1 (Parp-1)-deficient (Parp-1^−/−) mice. (a) Splenic B cells were stained with anti-B220, anti-immunoglobulin M (IgM) and anti-IgD. Live-gated, B220-expressing cells were counted. There were similar numbers of IgM⁻IgD⁻ cells. (b) Splenic B cells were stained with anti-B220, anti-CD21 and anti-CD23. Live-gated, B220-expressing cells were counted. Marginal zone cells (CD21⁺CD23^low) were increased in Parp-1^−/− mice, whereas naïve B cells (CD21^low CD23^low) were reduced. Follicle cell (CD21⁺CD23⁺) numbers were similar in Parp-1^−/− and wild-type mice. Experiments were carried out in four animals and similar results were obtained. (c) Comparison of absolute numbers of marginal zone, follicular and naïve B cells. Percentages were multiplied by the total number of splenic B cells to obtain absolute numbers of cells in the splenic B-cell subsets. The mean ± standard error of the mean from analysis of four mice is presented. There were significant differences in the numbers of marginal zone (P = 0·029; Mann–Whitney test) and naïve B cells (P = 0·029; Mann–Whitney test).

Antibody responses following intraperitoneal immunization. (a) Groups of six mice were immunized intraperitoneally with 100 μg of alum-precipitated NP-Ficoll (a T cell-independent antigen). Specific anti-NP immunoglobulin M (IgM) was measured by enzyme-linked immunosorbent assay (ELISA) after 12 days. There was no significant difference between wild-type and poly(ADP-ribose) polymerase-1 (Parp-1)-deficient (Parp-1^−/−) mice. Circles are pre-immune serum, and squares represent day 12 values. Closed symbols are wild-type (WT) and open symbols Parp-1-deficient animals. (b) Groups of six mice were immunized intraperitoneally with 100 μg of alum-precipitated NP-KLH (a T cell-dependent antigen). IgG1 NP-specific antibody responses of individual animals, 12 days after immunization, are shown. Circles are pre-immune serum, squares are day 12, and triangles are day 47 values. Filled symbols are wild-type animals and open symbols are Parp-1^−/− mice. (c) Specific antibody-secreting cells, from bone marrow-derived mononuclear cells, were counted by enzyme-linked immunosorbent spot-forming cell assay (ELISpot). Experiments were carried out in triplicate and from three individual knock-out and wild-type animals. The mean ± standard error of the mean is presented. Parp-1^−/− mice had reduced numbers of plasma cells secreting anti-NP (P = 0.02; Mann–Whitney test). (d) Frozen sections of spleens were stained with peanut agglutinin to detect germinal centres. The panels on the left are from Parp-1^−/− mice and those on the right are from wild-type mice. Germinal centres were of similar size in wild-type and Parp-1^−/− mice.