Long-lived bone marrow plasma cells are induced early in response to T cell-independent or T cell-dependent antigens

Abstract

The signals required to generate long-lived plasma cells remain unresolved. One widely cited model posits that long-lived plasma cells derive from germinal centers (GCs) in response to T cell-dependent (TD) Ags. Thus, T cell-independent (TI) Ags, which fail to sustain GCs, are considered ineffective at generating long-lived plasma cells. However, we show that long-lived hapten-specific plasma cells are readily induced without formation of GCs. Long-lived plasma cells developed in T cell-deficient mice after a single immunization with haptenated LPS, a widely used TI Ag. Long-lived plasma cells also formed in response to TD Ag when the GC response was experimentally prevented. These observations establish that long-lived plasma cells are induced in both TI and TD responses, and can arise independently of B cell maturation in GCs.

Figure 1. Kinetics of the plasma cell response after NP-LPS immunization

(A) NP-specific λ⁺ plasma cells (PCs) in the spleen and BM were quantified by ELISPOT after B6 adults were immunized once with NP-LPS in PBS. (B) B6 (circles, solid line) or B6.TcRβ^−/−δ^−/− (squares, dashed line) adults were immunized and evaluated as in (A). For (A) and (B) all symbols represent data from an individual mouse. Note: On day 91 BM PC frequencies were significantly different between B6 and B6.TcRβ^−/−δ^−/− mice as indicated by the * (p=0.002). At all other times points there were no significant differences in PC frequencies between these groups. (C) Representative ELISPOT wells from the BM (d0, d90) or spleen (d3) of B6.TcRβ^−/−δ^−/− mice.

Figure 2. Early NP-specific plasma cells are radioresistant

(A) Flow cytometric approach for sorting NP-specific CD138⁺ plasma cells (PCs) from spleens of mice immunized 7 days previously with NP-CGG/alum. Cells from the CD138⁺ gate were sorted for subsequent irradiation experiments. In addition to NP-binding plasma cells, radio-sensitivity was also determined for naive IgD⁺ NP⁻ B220⁺ CD19⁺ B cells sorted from alum and NP-CGG immunized mice (gate not shown). (B) Alum or NP-CGG/alum immunized mice were used as donors for the indicated cell populations including NP-binding CD138⁺ plasma cells. Sorted cells were either rested (top) or subjected to 200R (bottom) before resting. Nine hours later viability was determined by flow cytometry using DAPI. (C) Frequencies of cells secreting NP-specific antibodies were determined by ELISPOT using freshly sorted (open), sorted and cultured for 9 hours (black), or sorted, exposed to 200R IR, and then rested for 9 hours (gray) CD138⁺ NP-binding cells. (D) Separate experiment in which B6 adults immunized with NP-LPS in PBS or NP-CGG in alum were sacrificed on day 3 post-immunization. Single splenocyte suspensions were exposed to 0, 200, 600, or 800 rads, rested 9 hours, and then transferred to NP-coated ELISPOT plates to assay frequencies of NP-specific λ⁺ PCs. Non-irradiated samples are shown with closed symbols; irradiated samples with open symbols. Differences in PC frequencies in C and D were not statistically significant.

Figure 3. Decay rate for NP-LPS induced plasma cells

Cohorts of NP-LPS immunized B6 adults were left untouched or irradiated/reconstituted as described in Materials and Methods. (A) Shown are ELISPOT results for NP-specific λ⁺ plasma cells (PCs) in the BM comparing non-irradiated controls (circles with solid line) versus irradiated (squares with dashed line) B6 mice. Lines are drawn through the mean for each group. On day 190 BM PC frequencies were significantly different between irradiated and control mice as indicated by the * (p=0.05). At all other times points there were no significant differences in PC frequencies between these groups. (B) The decay rate NP-LPS induced PCs was illustrated by dividing the number of NP-specific PCs in each irradiated mouse by the average number of NP-specific PCs in 3–5 controls for each indicated time point as previously described by Slifka et al (6). The half-life (t_1/2) of BM plasma cells post-IR was calculated as follows: t_1/2 = (T × log 2) / log (starting quantity / ending quantity) where T= elapsed time.

Figure 4. Pulse-chase kinetics for hapten-specific NP-LPS induced plasma cells

NP-LPS immunized B6 adults were fed BrdU in the drinking water for 3 days post-immunization. (A) The frequency of NP-binding splenic plasma cells that were BrdU⁺ on day 3 post-immunization is shown. Data are the mean of four mice. (B) Frequencies of NP-binding BM plasma cells that were BrdU⁺ on days 34, 64, and 94 days of the chase are shown. Each circle represents an individual mouse, and the mean for each time point is indicated by a solid line. The half-life (t_1/2) of BM plasma cells post-IR was calculated as described for Figure 3. (C) Representative flow cytometric data illustrating the identification of NP-binding BrdU^+/− plasma cells in the BM at the indicated time points of the chase period. Note that BrdU⁺ cells were identified by comparing immunized controls that were not exposed to BrdU. The “Dump” channel includes antibodies to CD4, CD8, F4/80, TER-119 and Gr-1. Each plots was derived from files containing 7–9 × 10⁶ events.

Figure 5. Failure to detect GCs in immunized T cell deficient mice

B6 or B6.TcRβ^−/−δ^−/− adults were immunized with either NP-LPS/PBS or NP-CGG/alum, and frequencies of NP-binding CD19⁺ PNA^high GC B cells in the spleen determined by flow cytometry. (A) Flow cytometric data for the indicated mice, representative of 3–6 mice per group. Left-most plots are pre-gated on viable (DAPI⁻) IgD⁻ cells. 10⁶ events were collected for each sample. Antibodies used for the “Dump” channel are as described for Figure 4. (B) Numbers of NP-specific GC B cells at 3, 5, or 7 days were calculated by multiplying the frequency of hapten-binding GC B cells using the gating strategy shown in (A) by the total number of splenocytes harvested in each mouse. Results are expressed as means +/− SEM of 3–6 mice per group performed over two separate experiments. Absolute numbers for days 3, 5, and 7 are graphed separately to better reveal the small number of PNA-binding B cells in NP-LPS immunized mice at early time points, which fell below detection levels in all mice after day 5.

Figure 6. Long-lived T-cell dependent plasma cells without maturation in GCs

(A) Frequencies of NP-specific λ⁺ plasma cells (PCs) post-IR in the BM of B6 adults immunized with NP-CGG/alum and then left untouched (closed) or irradiated and reconstituted (open) on day 5 post-immunization (see Methods). (B) BM cells from the mice in (A) on day 105 post-IR were assayed by ELISPOT using either NP₃₃-BSA coated plates evaluated with anti-IgM specific antibodies (left) or NP₄-BSA coated plated evaluated with anti-IgG1 specific antibodies. Differences in λ⁺ and IgM⁺ PC frequencies between irradiated and control groups were not significant. The p value for differences in frequencies of NP-specific IgG1⁺ PCs is shown on the graph. (C, D) NP-CGG/alum immunized B6 adults were given three injections of anti-CD154 (MR-1) antibodies (open) or hamster IgG (closed) beginning 5 days post-immunization (see Methods). (C) Shown are BM NP-specific λ⁺ PCs assayed with NP₃₃-BSA. Differences between MR1 treated and control mice were significantly different on day 14 (p= 0.02) and day 190 (p=0.04). At all other times points there were no significant differences between these groups. (D) NP-specific IgM⁺ (left) PCs were assayed with NP₃₃-BSA (left) or NP-specific IgG1⁺ PCs were evaluated with NP₄-BSA (right). p values for statistically significant differences are shown on graphs. All results are expressed as means +/− SEM of 3–4 mice per group.