CD40-Activated B Cells Can Efficiently Prime Antigen-Specific Naïve CD8+ T Cells to Generate Effector but Not Memory T cells

Background: The identification of the signals that should be provided by antigen-presenting cells (APCs) to induce a CD8 + T cell response in vivo is essential to improve vaccination strategies using antigen-loaded APCs. Although dendritic cells have been extensively studied, the ability of other APC types, such as B cells, to induce a CD8 + T cell response have not been thoroughly evaluated. Methodology/Principal Findings: In this manuscript, we have characterized the ability of CD40-activated B cells, stimulated or not with Toll-like receptor (TLR) agonists (CpG or lipopolysaccharide) to induce the response of mouse naïve CD8 + T cells in vivo. Our results show that CD40-activated B cells can directly present antigen to naïve CD8 + T cells to induce the generation of potent effectors able to secrete cytokines, kill target cells and control a Listeria monocytogenes infection. However, CD40-activated B cell immunization did not lead to the proper formation of CD8 + memory T cells and further maturation of CD40-activated B cells with TLR agonists did not promote the development of CD8 + memory T cells. Our results also suggest that inefficient generation of CD8 + memory T cells with CD40-activated B cell immunization is a consequence of reduced Bcl-6 expression by effectors and enhanced contraction of the CD8 + T cell response. Conclusions: Understanding why CD40-activated B cell immunization is defective for the generation of memory T cells and gaining new insights about signals that should be provided by APCs are key steps before translating the use of CD40-B cel

CD40-B cell immunization generates effectors expressing similar level of T-bet and Blimp-1, higher level of Eomes and lower amount of Bcl-6.

<p>A. Expression of T-bet, Eomes and Bcl-6 by CD8⁺ Te cells generated following CD40-B cell and DC immunizations. Four days post-immunization with 2×10⁶ CD40-B cells or DCs matured with LPS and loaded with the OVA peptide, Te cells were stained intracellularily with antibodies against T-bet, Eomes and Bcl-6 transcription factors. The representative overlay histogram shows expression of the transcription factor by endogenous T cells (CD8⁺CD45.2⁻) and OVA-specific Te cells (CD8⁺CD45.2⁺). The MFI is shown on each overlay, the upper bold number indicates the MFI of OVA-specific effectors (CD8⁺CD45.2⁺) while the lower number is for the endogenous population (CD8⁺CD45.2⁻). B. Quantification of the level of expression of T-bet, Eomes and Bcl-6. The histograms shows the MFI of expression for T-bet, Eomes and Bcl-6 by OVA-specific CD8⁺ Te cells (CD8⁺CD45.2⁺) normalized to the MFI of endogenous CD8⁺ T cells (CD8⁺CD45.2⁻). The results are from at least 2 independent experiments. C. Similar expression of Blimp-1 by OVA-specific Te cells following CD40-B cell or DC immunization. At the peak of the response (d4), Te cells were sorted (CD8⁺CD45.2⁺) from spleen to extract RNA. The relative expression of Blimp-1 was determined by quantitative RT-PCR. Expression relative to a reference sample is shown. Results are from 4 independent experiments. * p<0.05 and *** p<0.001.</p

CD40-B cells have an activated phenotype.

<p>After 3 d of culture on murine 3T3-CD40L fibroblasts, CD40-B cells were matured or not with LPS (1 µg/mL) or CpG (2 mM) for 24 h (CD40-B LPS and CD40-B CpG). Freshly isolated splenocytes were used as a naïve B cell control. The histogram bars show the mean of fluorescence intensity (MFI) +/− standard deviation of the mean (SEM) for the expression of CD86, CD80, CD62L, CD40, K^b and I-A^b gated on the CD19⁺ population. The results are pooled from at least three independent experiments except for CD40 expression on B cells (n = 2). * p<0.05, ** p<0.01 and *** p<0.001.</p

Immunization with CD40-B cells induces an <i>in vivo</i> CD8⁺ T cell response.

<p>A. CD40-B cell vaccination generates CD8⁺ Te cells but not Tm cells. 10⁶ female OT-1 T cells (CD8⁺CD45.2⁺) were adoptively transferred into congenic B6SJL female mice (CD45.1⁺) followed by immunization two days later with 2×10⁶ CD40-B cells, matured or not with LPS (1 µg/mL) or CpG (2 mM) and loaded with 4 µg/mL OVA or with an irrelevant peptide (IRR). As a reference recipients were immunized with 2×10⁶ DCs matured with LPS and loaded with OVA peptide. OVA-specific T cells (CD8⁺CD45.2⁺) were analyzed in the same mouse by surgical removal of superficial lymph nodes at d4 (effector) and d30–45 (memory) post-immunization. Te and Tm cells were identified as CD8⁺CD45.2⁺ by flow cytometry. The percentage of Te and Tm cells generated are indicated on each dot plot. B. Percentage of CD8⁺ Te (left panel) and Tm (right panel) cells recovered at d4 (Te) and d>30 (Tm) in one lymph node is shown. C. Yield of CD8⁺ Tm cell generation. The yield of Tm cell formation was calculated as the percentage of Te cells that develop into Tm cells. D. The percentage of mice that generates more then 5% of CD8⁺ Tm cells is shown for the different immunization conditions. E and F. Lm challenges. 30 d post immunization, mice were challenged with a lethal dose of Lm-OVA (10⁵ CFU). 3 d post challenged, CFU were determined in the spleen (E) and liver (F) for each mouse. A–D are from at least four independent experiments with at least two mice per group while E and F are from one independent experiment with three mice per group. * p<0.05, ** p<0.01 and *** p<0.001.</p

Effectors generated with CD40-B cell immunization contract more rapidly than the one obtained with DC immunization.

<p>A. Contraction of the OVA-specific CD8⁺ T cell response. Mice were immunized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030139#pone-0030139-g002" target="_blank">Figure 2</a>. Lymph nodes were surgically removed at 4, 7, 10 and >30 days post-immunization. Cells were stained to determine the percentage of Te cells generated. The graph shows the percentage of remaining Te cells (CD8⁺CD45.2⁺) over time relative to the peak of the response (d4). B. Effectors generated with CD40-B cell and DC immunization express similar amount of Bcl-2 during the course of the CD8⁺ T cell response. The MFI of Bcl-2 for OVA-specific CD8⁺ Te cells was normalized to the MFI of endogenous CD8⁺ T cells to obtain a MFI ratio. 3 independent experiments. * p<0.05, ** p<0.01 and *** p<0.001.</p

CD40-B cell vaccination generates functional effector.

<p>A. <i>In vivo</i> killing. Mice were immunized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030139#pone-0030139-g002" target="_blank">Figure 2</a>. Four days post-immunization, CFSE-labeled splenocytes pulsed or not with OVA were injected as target cells. After 4 h, the percentage of CFSE^hi (OVA-pulsed; gate labeled OVA on the histogram) and CFSE^lo (unpulsed; gate labeled neg on the histogram) cells were analyzed in the spleen. Percentage of specific lysis is indicated on the histogram and was calculated using the indicated gate and as described in Material and Methods. B. Percentage of specific killing by OVA CD8⁺ Te cells. Mean +/− SEM of specific lysis are shown for the different immunization conditions. 2 mice per conditions, 3 independent experiments. C and D. Lm challenge. Four days post-immunization, mice were challenged with a lethal dose of Lm-OVA (10⁵ CFU). 3 d post-infection (peak of bacterial load), mice were killed and CFU were determined in the spleen (C) and the liver (D). Mean +/− standard (SD) are shown. 2–4 mice per conditions, 3 independent experiments. * p<0.05, ** p<0.01 and *** p<0.001.</p

Phenotype of the CD8⁺ Te cells generated after CD40-B cell immunization.

<p>A. Phenotype of effectors at d4 post-immunization with CD40-B cells treated or not with TLR ligands. Immunizations were realized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030139#pone-0030139-g002" target="_blank">Figure 2</a> with CD40-B cells (2×10⁶) treated or not with CpG or LPS and with DCs (2×10⁶) matured with LPS. The bar chart shows the MFI of expression for CD44, 1B11, CD127, CD122, CD27 by CD8⁺ Te cells (CD8⁺CD45.2⁺) normalized to the MFI of endogenous CD8⁺ T cells (CD8⁺CD45.2⁻). For CD62L, the percentage of CD8⁺ Te cells expressing high level of CD62L is shown. B. No correlation between CD127 expression level and memory generation. The results are from 2 independent experiments for CD27 and CD122 and from at least 3 independent experiments for the other cell surface molecules. * p<0.05, ** p<0.01 and *** p<0.001.</p