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Regulation and Maintenance of an Adoptive T-Cell Dependent Memory B Cell Pool

View Article: PubMed Central - PubMed

ABSTRACT

We investigated the ability of monoclonal B cells to restore primary and secondary T-cell dependent antibody responses in adoptive immune-deficient hosts. Priming induced B cell activation and expansion, AID expression, antibody production and the generation of IgM+IgG- and IgM-IgG+ antigen-experienced B-cell subsets that persisted in the lymphopenic environment by cell division. Upon secondary transfer and recall the IgM-IgG+ cells responded by the production of antigen-specific IgG while the IgM+ memory cells secreted mainly IgM and little IgG, but generated new B cells expressing germinal center markers. The recall responses were more efficient if the antigenic boost was delayed suggesting that a period of adaptation is necessary before the transferred cells are able to respond. Overall these findings indicate that reconstitution of a functional and complete memory pool requires transfer of all different antigen-experienced B cell subsets. We also found that the size of the memory B cell pool did not rely on the number of the responding naïve B cells, suggesting autonomous homeostatic controls for naïve and memory B cells. By reconstituting a stable memory B cell pool in immune-deficient hosts using a monoclonal high-affinity B cell population we demonstrate the potential value of B cell adoptive immunotherapy.

No MeSH data available.


Related in: MedlinePlus

Functions of AID/YFP+ memory B cell subsets.Rag2-/- recipient mice were injected with naive SWHEL.AID/YFP.Rag2-/- B cells and OTII.Rag2-/- naive CD4 T cells and immunized as in Fig 1. Eight weeks after immunization, AID/YFP+ IgM+ and AID/YFP+ IgG+ subsets were isolated and transferred alone into secondary Rag2-/- recipient mice. Naive B cells were transferred as controls. (A) Numbers of splenic B cells recovered from the different secondary hosts without immunization. (B) The secondary hosts were immunized 1 day after transfer and the seric levels of anti-HEL specific IgG (C) and IgM were measured 6, 9, 12, 16, 20 and 30 days after immunization. (D) the % of splenic Gl7 hi B cells in secondary hosts transferred with YFP+ IgM+ (white) or YFP+ IgG+ (black) B cells was analyzed by Flow Cytometry 3 weeks after immunization and (F) the relative % of AID/YFP- and AID/YFP+ cells was assessed. In another settings, (G) the different secondary hosts were immunized either 1 day (black circles) or 30 days (white circles) after transfer and the number of total splenic B cells recovered from the spleen of different secondary hosts was determined 6 weeks after immunization. Data (mean ± SEM) are shown for one experiment representative of 2, with 4–5 mice per group. Significances were calculated using Student t-tests, *, P<0.05; ***, P<0.001.
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pone.0167003.g005: Functions of AID/YFP+ memory B cell subsets.Rag2-/- recipient mice were injected with naive SWHEL.AID/YFP.Rag2-/- B cells and OTII.Rag2-/- naive CD4 T cells and immunized as in Fig 1. Eight weeks after immunization, AID/YFP+ IgM+ and AID/YFP+ IgG+ subsets were isolated and transferred alone into secondary Rag2-/- recipient mice. Naive B cells were transferred as controls. (A) Numbers of splenic B cells recovered from the different secondary hosts without immunization. (B) The secondary hosts were immunized 1 day after transfer and the seric levels of anti-HEL specific IgG (C) and IgM were measured 6, 9, 12, 16, 20 and 30 days after immunization. (D) the % of splenic Gl7 hi B cells in secondary hosts transferred with YFP+ IgM+ (white) or YFP+ IgG+ (black) B cells was analyzed by Flow Cytometry 3 weeks after immunization and (F) the relative % of AID/YFP- and AID/YFP+ cells was assessed. In another settings, (G) the different secondary hosts were immunized either 1 day (black circles) or 30 days (white circles) after transfer and the number of total splenic B cells recovered from the spleen of different secondary hosts was determined 6 weeks after immunization. Data (mean ± SEM) are shown for one experiment representative of 2, with 4–5 mice per group. Significances were calculated using Student t-tests, *, P<0.05; ***, P<0.001.

Mentions: Memory B cells are defined functionally by their ability to induce secondary IgG antibody responses upon secondary antigenic challenge. We investigated whether the subsets of AID/YFP+IgM+ and AID/YFP+IgM-IgG+ antigen-experienced (memory) B cells persisting at late time points could mount secondary IgG responses and persist after secondary transfer. For this purpose we followed two different experimental strategies. In the first, 5x104 cells of either IgM+ or IgM-IgG+ memory B cells, were transferred with an excess helper OTII CD4+ T cells into secondary Rag-deficient hosts that were boosted with OVA-HEL the day after cell transfer. In the absence of immunization antibody levels were undetectable (not shown) and three weeks after transfer recovery of both memory B cell subsets was about 10–20% of the initial cell input, exceeding naïve B cell recovery (Fig 5A), supporting the notion that memory B cells may not require specific ligand recognition to survive (2). One cannot exclude, however, that cross-reactivity of the BCR transgene with environmental antigens may allow signaling sufficient to maintain naïve and memory cell survival in the absence of HEL [24]. Following immunization, the secondarily transferred AID/YFP+IgM-IgG+ cells responded promptly with the exclusive production of significant levels HEL-specific IgG thus confirming their memory statute (11). The AID/YFP+IgM+ B cells in response to antigenic boost produced only limited amounts of IgM antibodies (Fig 4B), little IgG antibodies, but did generate GL7+ B cells more efficiently than the IgG+ memory B cell population (Fig 5D). Thus the IgM+ subset may contain precursors able to generate a secondary germinal center reaction and a new progeny of IgG+ effectors (4). With time antibody levels decayed rapidly suggesting that the number of transferred memory B cells declined in the secondary hosts after antigenic boost. Indeed, IgM+ and IgG+ memory B cells failed to expand and 3 weeks after immunization cell recovery was similar to the retrieval observed in the non-immunized hosts (compare Fig 5E and 5A). In similar experimental conditions, naïve B cells following immunization expanded, acquired AID/YFP expression and their numbers more than doubled the number initially injected (Figs 5F and 2A). These data suggest that a significant fraction of the memory B cells generated have a reduced expansion capacity being programmed for rapid differentiation for effector functions.


Regulation and Maintenance of an Adoptive T-Cell Dependent Memory B Cell Pool
Functions of AID/YFP+ memory B cell subsets.Rag2-/- recipient mice were injected with naive SWHEL.AID/YFP.Rag2-/- B cells and OTII.Rag2-/- naive CD4 T cells and immunized as in Fig 1. Eight weeks after immunization, AID/YFP+ IgM+ and AID/YFP+ IgG+ subsets were isolated and transferred alone into secondary Rag2-/- recipient mice. Naive B cells were transferred as controls. (A) Numbers of splenic B cells recovered from the different secondary hosts without immunization. (B) The secondary hosts were immunized 1 day after transfer and the seric levels of anti-HEL specific IgG (C) and IgM were measured 6, 9, 12, 16, 20 and 30 days after immunization. (D) the % of splenic Gl7 hi B cells in secondary hosts transferred with YFP+ IgM+ (white) or YFP+ IgG+ (black) B cells was analyzed by Flow Cytometry 3 weeks after immunization and (F) the relative % of AID/YFP- and AID/YFP+ cells was assessed. In another settings, (G) the different secondary hosts were immunized either 1 day (black circles) or 30 days (white circles) after transfer and the number of total splenic B cells recovered from the spleen of different secondary hosts was determined 6 weeks after immunization. Data (mean ± SEM) are shown for one experiment representative of 2, with 4–5 mice per group. Significances were calculated using Student t-tests, *, P<0.05; ***, P<0.001.
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pone.0167003.g005: Functions of AID/YFP+ memory B cell subsets.Rag2-/- recipient mice were injected with naive SWHEL.AID/YFP.Rag2-/- B cells and OTII.Rag2-/- naive CD4 T cells and immunized as in Fig 1. Eight weeks after immunization, AID/YFP+ IgM+ and AID/YFP+ IgG+ subsets were isolated and transferred alone into secondary Rag2-/- recipient mice. Naive B cells were transferred as controls. (A) Numbers of splenic B cells recovered from the different secondary hosts without immunization. (B) The secondary hosts were immunized 1 day after transfer and the seric levels of anti-HEL specific IgG (C) and IgM were measured 6, 9, 12, 16, 20 and 30 days after immunization. (D) the % of splenic Gl7 hi B cells in secondary hosts transferred with YFP+ IgM+ (white) or YFP+ IgG+ (black) B cells was analyzed by Flow Cytometry 3 weeks after immunization and (F) the relative % of AID/YFP- and AID/YFP+ cells was assessed. In another settings, (G) the different secondary hosts were immunized either 1 day (black circles) or 30 days (white circles) after transfer and the number of total splenic B cells recovered from the spleen of different secondary hosts was determined 6 weeks after immunization. Data (mean ± SEM) are shown for one experiment representative of 2, with 4–5 mice per group. Significances were calculated using Student t-tests, *, P<0.05; ***, P<0.001.
Mentions: Memory B cells are defined functionally by their ability to induce secondary IgG antibody responses upon secondary antigenic challenge. We investigated whether the subsets of AID/YFP+IgM+ and AID/YFP+IgM-IgG+ antigen-experienced (memory) B cells persisting at late time points could mount secondary IgG responses and persist after secondary transfer. For this purpose we followed two different experimental strategies. In the first, 5x104 cells of either IgM+ or IgM-IgG+ memory B cells, were transferred with an excess helper OTII CD4+ T cells into secondary Rag-deficient hosts that were boosted with OVA-HEL the day after cell transfer. In the absence of immunization antibody levels were undetectable (not shown) and three weeks after transfer recovery of both memory B cell subsets was about 10–20% of the initial cell input, exceeding naïve B cell recovery (Fig 5A), supporting the notion that memory B cells may not require specific ligand recognition to survive (2). One cannot exclude, however, that cross-reactivity of the BCR transgene with environmental antigens may allow signaling sufficient to maintain naïve and memory cell survival in the absence of HEL [24]. Following immunization, the secondarily transferred AID/YFP+IgM-IgG+ cells responded promptly with the exclusive production of significant levels HEL-specific IgG thus confirming their memory statute (11). The AID/YFP+IgM+ B cells in response to antigenic boost produced only limited amounts of IgM antibodies (Fig 4B), little IgG antibodies, but did generate GL7+ B cells more efficiently than the IgG+ memory B cell population (Fig 5D). Thus the IgM+ subset may contain precursors able to generate a secondary germinal center reaction and a new progeny of IgG+ effectors (4). With time antibody levels decayed rapidly suggesting that the number of transferred memory B cells declined in the secondary hosts after antigenic boost. Indeed, IgM+ and IgG+ memory B cells failed to expand and 3 weeks after immunization cell recovery was similar to the retrieval observed in the non-immunized hosts (compare Fig 5E and 5A). In similar experimental conditions, naïve B cells following immunization expanded, acquired AID/YFP expression and their numbers more than doubled the number initially injected (Figs 5F and 2A). These data suggest that a significant fraction of the memory B cells generated have a reduced expansion capacity being programmed for rapid differentiation for effector functions.

View Article: PubMed Central - PubMed

ABSTRACT

We investigated the ability of monoclonal B cells to restore primary and secondary T-cell dependent antibody responses in adoptive immune-deficient hosts. Priming induced B cell activation and expansion, AID expression, antibody production and the generation of IgM+IgG- and IgM-IgG+ antigen-experienced B-cell subsets that persisted in the lymphopenic environment by cell division. Upon secondary transfer and recall the IgM-IgG+ cells responded by the production of antigen-specific IgG while the IgM+ memory cells secreted mainly IgM and little IgG, but generated new B cells expressing germinal center markers. The recall responses were more efficient if the antigenic boost was delayed suggesting that a period of adaptation is necessary before the transferred cells are able to respond. Overall these findings indicate that reconstitution of a functional and complete memory pool requires transfer of all different antigen-experienced B cell subsets. We also found that the size of the memory B cell pool did not rely on the number of the responding na&iuml;ve B cells, suggesting autonomous homeostatic controls for na&iuml;ve and memory B cells. By reconstituting a stable memory B cell pool in immune-deficient hosts using a monoclonal high-affinity B cell population we demonstrate the potential value of B cell adoptive immunotherapy.

No MeSH data available.


Related in: MedlinePlus