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

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

Functions of AID/YFP+ memory B cell subsets.

<p>Rag2-/- recipient mice were injected with naive SW_HEL.AID/YFP.Rag2^-/- B cells and OTII.Rag2-/- naive CD4 T cells and immunized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167003#pone.0167003.g001" target="_blank">Fig 1</a>. 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 <i>t</i>-tests, *, P<0.05; ***, P<0.001.</p

Regulation of the size of the memory B cell pool.

<p>Rag2-/- recipient mice were injected with different numbers of naive SW_HEL.AID/YFP.Rag2^-/- B cells and OTII.Rag2-/- naive CD4 T cells and immunized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167003#pone.0167003.g001" target="_blank">Fig 1</a>. The correlations between the number of injected B cells and (A) the number of total splenic B cells recovered, (B) the number of splenic AID/YFP+ memory B cells recovered 8 weeks after immunization and (C) the level of anti-HEL specific IgG in the serum are shown. For each plot, linear regression coefficient R2 is shown.</p

Endogenous TCR Recombination in TCR Tg Single RAG-Deficient Mice Uncovered by Robust In Vivo T Cell Activation and Selection

International audienceRecombination activating gene (RAG)-deficient TCR (T Cell Receptor) Tg (transgenic) mice are routinely used as sources of monoclonal T cells. We found that after the transfer of T cells from a RAG-2-deficient 5CC7 TCR Tg mice into allogeneic hosts we recovered a population of T cells expressing diverse αβ-TCRs. In fact, in the thymus and spleen of the 5CC7 RAG-2-deficient donor mice, we detected rare T cells expressing non-Tg TCR chains. Similar observations were obtained using T cells from two other TCR transgenic strains, namely RAG-2-deficient aHY and RAG-1-deficient OT-1 mice. The sequences of the endogenous TCR transcripts suggested that gene recombination could occur, albeit quite inefficiently, in the RAG-deficient mice we used. In agreement, we evidenced rare TCR Vα and Vβ-chain transcripts in non-Tg RAG-2-deficient mice. Since in these non-Tg RAG-deficient mice no mature T cells could ever be found, our findings suggested a role for the TCR Tg in rescuing rare recombined endogenous chains. Robust T-cell activation by the allogeneic environment favored the selection and expansion of the rare cells expressing endogenous TCRs. Potential mechanisms involved in the recombination of the endogenous TCR chains in the different strains of RAG-deficient mice used, and in particular the possibility of RAG-1 hypomorphism due to an incomplete knocking out procedure, are discussed. Our findings have important experimental implications for studies using TCR-Tg RAG-deficient cells as monoclonal T cell populations

Transcriptional program of memory B cell subsets.

<p>Transcriptional program of memory B cell subsets.</p

Development of B cell primary response upon adoptive transfer of SW_HEL.AID/YFP.Rag2^-/- B cells.

<p>(A) Rag2-/- hosts were injected intravenously with naive SW_HEL.AID/YFP.Rag2^-/- B cells together with OTII.Rag2^-/- naive CD4 T cells. Recipient mice were immunized 24 hours later and B cell response analyzed 7, 14 days and 8 weeks after. (B) Flow cytometric analyses of specific HEL⁺ CD19⁺ (left panels) splenic B cells from SW_HEL.AID/YFP.Rag2^-/- naive donor (upper panels), immunized mice reconstituted only with B cells (middle panels) and immunized mice reconstituted with both B and T cells 14 days after immunization (lower panels), for the expression of YFP (middle panels), and IgM and IgG (right panels). (C) Number of splenic B cells recovered from the recipient mice (left panel) and repartition of AID/YFP- and AID/YFP+ among splenic B cells (right panel) 7, 11 and 14 days after immunization. (D) Seric levels of anti-HEL specific IgG (left panel) and IgM (right panel) in immunized recipients 7, 11 and 14 days after immunization. (E) AID/YFP (green), B220 (red), IgD (blue) (right panels) and merged <b>(</b>left and right panels) expression by confocal microscopy analysis of spleen slices 14 days after immunization. (F) Analysis of germinal center B cells in immunized mice. Left panel shows the co-expression of Gl7 and CD95 by B cells from naive (left dot plot), non-immunized (middle dot plot) and 14 day-immunized mice (right dot plot). Right panel shows the % of Gl7hi CD95+ cells among AID/YFP- (white bar) and AID/YFP+ (black bar) splenic B cell subsets from 14 day-immunized mice. Data (mean ± SEM) are shown for one experiment representative of 3, with 4–5 mice per group. Significances were calculated using Student <i>t</i>-tests, *, P<0.05; ***, P<0.001.</p

Long-term maintenance of memory B cells.

<p>Rag2-/- recipient mice were injected with naive SW_HEL.AID/YFP.Rag2^-/- B cells and OTII.Rag2-/- naive CD4 T cells and immunized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167003#pone.0167003.g001" target="_blank">Fig 1</a>. Mice were analyzed at 2, 4, 8, 12 and 20 weeks after immunization. (A) Number of splenic B cells recovered. (B) Seric level of anti-HEL specific IgG1. (C) Number of splenic CD138+ plasma cells determined by flow cytometry. (D) Repartition of splenic AID/YFP+ (black bars) and AID/YFP- (white bars) B cells. (E) Flow cytometric analyses of IgG and IgM expression by AID/YFP+ B cells. (F) Flow cytometric analyses of MHC II, CD80, CD95, CXCR5 and Gl7 expression by splenic AID/YFP+ (green lines), AID/YFP- (blue lines) and naive (black lines, upper histograms), and IgM+ (black lines) and IgG+ (red lines, lower histograms) B cells from naive, and eight-week immunized mice. (G) The % of splenic proliferating B cell was assessed 8 weeks after immunization by flow cytometric analysis of Ki67 expression (left) and BrdU incorporation 24h (middle) or 72h (right) after BrdU injection. (H) The % of apoptotic splenic B cell was assessed by flow cytometric analysis of active caspase 3 level 8 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 <i>t</i>-tests, *, P<0.05; ***, P<0.001.</p

In vivo and in absence of a thymus, the enforced expression of the Notch ligands delta-1 or delta-4 promotes T cell development with specific unique effects

The role of Notch signaling in T cell commitment during lymphoid development is well established. However, the identity of the ligand that triggers this critical signal in vivo is still unclear. By overexpressing Delta-1 and Delta-4 ligands in the hemopoietic cells of athymic nu/nu host mice, we demonstrate that, in vivo and in the absence of a thymus, Delta-1 or Delta-4 expression is sufficient to promote T cell development from the most immature progenitor stages to complete maturation of both CD8(+) and CD4(+) alphabeta T cells. The mature T cells developing in a Delta-1- or Delta-4-enriched environment express a diverse TCR repertoire, are able to proliferate upon in vitro TCR stimulation, but show different profiles of cytokine production after in vitro anti-CD3 stimulatio