BLyS inhibition eliminates primary B cells but leaves natural and acquired humoral immunity intact - PubMed (original) (raw)

. 2008 Oct 7;105(40):15517-22.

doi: 10.1073/pnas.0807841105. Epub 2008 Oct 1.

Jenni E Crowley, Mary M Tomayko, Natalie Steinel, Patrick J O'Neill, William J Quinn 3rd, Radhika Goenka, Juli P Miller, Yun Hee Cho, Vatana Long, Chris Ward, Thi-Sau Migone, Mark J Shlomchik, Michael P Cancro

Affiliations

BLyS inhibition eliminates primary B cells but leaves natural and acquired humoral immunity intact

Jean L Scholz et al. Proc Natl Acad Sci U S A. 2008.

Abstract

We have used an inhibiting antibody to determine whether preimmune versus antigen-experienced B cells differ in their requisites for BLyS, a cytokine that controls differentiation and survival. Whereas in vivo BLyS inhibition profoundly reduced naïve B cell numbers and primary immune responses, it had a markedly smaller effect on memory B cells and long-lived plasma cells, as well as secondary immune responses. There was heterogeneity within the memory pools, because IgM-bearing memory cells were sensitive to BLyS depletion whereas IgG-bearing memory cells were not, although both were more resistant than naïve cells. There was also heterogeneity within B1 pools, as splenic but not peritoneal B1 cells were diminished by anti-BLyS treatment, yet the number of natural antibody-secreting cells remained constant. Together, these findings show that memory B cells and natural antibody-secreting cells are BLyS-independent and suggest that these pools can be separately manipulated.

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Conflict of interest statement

Conflict of interest statement: M.P.C. was supported in part by a sponsored research agreement between Human Genome Sciences, Inc., and the University of Pennsylvania.

Figures

Fig. 1.

Fig. 1.

BLyS inhibition in vivo. (A) C57BL/6 mice were treated with 100 μg of anti-BLyS i.p. on days 0 and 5 and subsequently analyzed for serum BLyS (open bars) and anti-BLyS (filled bars). Each bar represents the average ± SD for at least three mice per time point. Untreated mice (n = 34) are shown at day 0. Combined data from three separate experiments are shown. (B) Numbers of splenic follicular (FO) B cells ± SD for the same mice shown in A.

Fig. 2.

Fig. 2.

Effects of BLyS inhibition on B1 subsets. (A) Gating scheme for identification of B2, B1a, and B1b cells in spleen (Upper) or peritoneal cavity (Lower). Lymphocyte-gated cells are further identified as B1a (CD5+ IgM+) or B2 (CD5− IgM+) as shown in left plots and histograms; in addition, B1a cells are CD23− (data not shown), whereas B2s are CD23+ and thus include late TR and FO B cells. B1b cells are CD5lo/− CD23− IgM+ Mac1+ as shown in right plots. (B) Numbers of splenic (Upper) and PerC (Lower) B2 and B1 cells. Cell numbers ± SD for three untreated mice (open bars) and three treated mice are shown. n.s., not significant. (C) Numbers of PC-specific antibody-producing cells in spleen and PerC of isotype control-treated (open symbols) or anti-BLyS-treated (filled symbols) mice at 10 and 21 days after treatment. Each symbol represents one mouse.

Fig. 3.

Fig. 3.

BLyS ablation does not alter NP-reactive memory B cells or LLPC generated in wild-type mice (Memory System 1). (A and B) Memory B cell analysis of C57BL/6 mice immunized with NP-CGG. Primary immunization was at day 0, anti-BLyS treatment was at day 56, and NP booster immunization was at day 77. Memory B cell numbers and IgG titers were assessed at day 84 for all mice including untreated and primed-only controls. NP-reactive memory B cells were identified according to the FACS gating scheme shown in A. After doublet discrimination, DAPI exclusion, and lymphocyte gating, memory B cells were phenotyped as CD4−CD8−Gr-1−B220+NP+. NT, not treated control. NP-reactive memory B cell numbers and high-affinity anti-NP IgG1 antibody titers, as assessed by ELISA, are shown in B. Each symbol in graphs represents an individual mouse, black lines represent means, and combined results from two separate experiments are shown. Symbols at the bottom indicate treatment given (+) or not given (−). (C) Number of NP-specific LLPC in the bone marrow of control mice or anti-BLyS-treated mice at 21 days after anti-BLyS treatment and 77 days after NP-CGG immunization. Phenotyping strategy for LLPC is presented in

Fig. S4

.

Fig. 4.

Fig. 4.

Memory B cells generated in adoptive transfer systems are more resistant to BLyS depletion than naïve B cells. Memory B cells were generated from mVh186.2 Tg (Memory System 2; A and B) or Vh186.2 KI (Memory System 3; C, E, and F) donor B cells after adoptive transfer into AM14/Vκ8R double Tg mice and subsequent immunization i.p. with NP-CGG in alum. Naïve controls were unimmunized Vh186.2 KI donor strain (D and E) and recipient IgMa non-NIP-binding (A–C and E) B cells. Recipient mice >12 weeks after immunization and unimmunized donor strain controls were treated with two 100-μg i.p. doses of anti-BLyS (10F4) or hamster IgG isotype control (IC). Fifteen days later, spleens were harvested and analyzed by FACS. (A) Memory System 2. Single, live lymphocytes were identified by FSC and SSC profiles and EMA exclusion. Upper and Lower represent isotype control (IC) versus anti-BLyS-treated animals, respectively. NIP+ IgMa+ B cells were NP-specific donor-derived memory cells. NIP− IgMa+ B cells were recipient AM14/Vκ8R specificity naïve B cells. IgMa− IgMb+ B cells were non-Tg-bearing recipient B cells. This population is expanded in aged recipient mice and may represent endogenous memory B cells. Representative FACS plots are shown. (B) Total numbers of B cells in each subgroup were calculated from live splenocyte counts and FACS-based frequencies as shown in A. Each point represents an individual animal. Average fold depletion in anti-BLyS-treated (circles) versus isotype control-treated (triangles) animals is indicated. One-way Student's t tests were performed. n.s., not significant. **, P ≤ 0.01; ***, P < 0.001. (C) Memory System 3. Live lymphocytes were identified as in A. NIP+ κlow B cells were NP-specific donor-derived memory cells. NIP− B cells were recipient-derived naïve cells. Memory B cells were fractionated into IgG1-switched and nonswitched B cells. (D) Naïve controls for Memory System 3. Spleens were harvested from unimmunized Vh186.2 KI donor-strain mice. Live lymphocytes were gated as described in A. (E and F) Total numbers of B cells from each System 3 subgroup were calculated from live splenocyte counts and FACS-based frequencies.

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