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

Natural Killer cell receptors and decreased susceptibility to HIV infection

The human immunodeficiency virus (HIV) currently infects over 33 million individuals worldwide. The development of a protective HIV vaccine would provide the ideal tool to fight this pandemic. Recent clinical trials testing candidate T- and B-cell based vaccine strategies have failed to demonstrate that these were protective against infection. For many, these failures underline how little is known about what constitutes an efficient immune response against HIV and that increased knowledge about the role of innate immune cells may be required in order to design an efficient vaccine against HIV.Natural Killer (NK) cells are part of the innate arm of the immune system and are involved in the control of several viral infections, including HIV. Epidemiological evidence has linked specific NK cell receptors, termed KIR3DS1 and KIR3DL1, to favourable clinical outcomes in HIV infected individuals. Whether these receptors would also be involved in protection from infection, and therefore could provide the basis for new vaccine strategies, is unknown.To address this question, we have evaluated the genetic distribution of both KIR3DS1 and KIR3DL1 in a population of exposed uninfected individuals (EUs). EUs remain HIV seronegative despite repeated exposure to the virus through high-risk behavior. Understanding the immunological causes of their decreased susceptibility to infection may provide insights into vaccine design. In chapter II we demonstrate that KIR3DS1 homozygous individuals are overrepresented in the EU population. Additionnally, in chapter III, we provide evidence that the combined genotype of HLA-B*57 with a specific set of KIR3DL1 alleles is also overrepresented in the EU population. Finally, in chapter IV, we demonstrate that NK cells from individuals carrying certain KIR3DL1/HLA genotypes linked to slower HIV disease progression and/or protection from infection have increased functional potential following stimulation.The evidence presented in this thesis supports a role for NK cells, and particularly of KIR3DS1 and KIR3DL1, in the decreased susceptibility to HIV infection observed in EUs. Given that these cells are directly involved in viral suppression and are capable of modulating both the adaptive and innate arm of the immune response, understanding how NK cell mediate these activities may reveal new therapeutic strategies against HIV.Le virus de l'immunodéficience humaine (VIH) infecte présentement plus de 33 millions d'individus. Contre cette pandémie, la solution idéale serait le développement d'un vaccin. Toutefois, de récents essais cliniques évaluant l'efficacité des stratégies de vaccination visant à la stimulation des cellules T ou B contre le VIH n'ont pas réussi à démontrer que ces stratégies protégeaient contre l'infection. Pour plusieurs membres de la communauté scientifique, ces échecs démontrent que les caractéristiques d'une réponse immunitaire efficace contre le VIH sont encore méconnues et que le rôle des cellules du système immun inné devra sans doute être éclairci afin d'élaborer un vaccin efficace contre le VIH.Les cellules NK (Natural Killer) font parties du système immunitaire inné et aident au contrôle de plusieurs infections virales, incluant les infections au VIH. Des études épidémiologiques ont lié certains récepteurs des cellules NK, appelés KIR3DS1 et KIR3DL1, à des indices cliniques favorables chez des individus infectés par le VIH. Que ces récepteurs puissent aussi pourvoir une forme de protection contre l'infection, et donc inspirer de nouvelles stratégies de vaccination, demeure encore incertain.Afin de répondre à cette question, nous avons évalué la distribution génétique de KIR3DS1 et KIR3DL1 dans une population d'individus exposés séronégatifs (ESN). Les ESN demeurent séronégatifs aux anticorps du VIH malgré des comportements à risques. Comprendre les facteurs immunitaires permettant à cette population d'être moins susceptible à l'infection au VIH pourrait aider à l'élaboration d'un vaccin. Lors du chapitre II, nous démontrons que les individus KIR3DS1 homozygotes sont surreprésentés dans la population d'ESN. De plus, dans le troisième chapitre, nos données soutiennent qu'une combinaison génétique du HLA-B*57 avec un sous-type particulier de KIR3DL1 et aussi surreprésentée dans la population d'ESN. Finalement, dans le chapitre IV, nous démontrons que les cellules NK provenant d'individus ayant des génotypes HLA/KIR3DL1 liés à une progression plus lente de la maladie VIH et/ou à une protection contre l'infection ont un potentiel de fonctionnalité accru suivant une stimulation.Les données présentées dans cette thèse suggèrent que les cellules NK, plus particulièrement leurs récepteurs KIR3DS1 et KIR3DL1, sont impliquées dans une forme de résistance à l'infection observée chez les ESN. Puisque ces cellules sont directement impliquées dans le contrôle viral et peuvent moduler à la fois le système immun inné et adapté, éclaircir les mécanismes permettant aux cellules NK de diminuer la susceptibilité à l'infection pourrait révéler de nouvelle stratégie thérapeutique afin de contrer le VIH

IL-6 Production by Dendritic Cells Is Dispensable for CD8+ Memory T-Cell Generation

Following activation, naïve CD8+ T cells will differentiate into effectors that differ in their ability to survive: some will persist as memory cells while the majority will die by apoptosis. Signals given by antigen-presenting cells (APCs) at the time of priming modulate this differential outcome. We have recently shown that, in opposition to dendritic cell (DC), CD40-activated B-(CD40-B) cell vaccination fails to efficiently produce CD8+ memory T cells. Understanding why CD40-B-cell vaccination does not lead to the generation of functional long-lived memory cells is essential to define the signals that should be provided to naïve T cells by APCs. Here we show that CD40-B cells produce very low amount of IL-6 when compared to DCs. However, supplementation with IL-6 during CD40-B-cell vaccination did not improve memory generation. Furthermore, IL-6-deficient DCs maintained the capacity to promote the formation of functional CD8+ effectors and memory cells. Our results suggest that in APC vaccination models, IL-6 provided by the APCs is dispensable for proper CD8+ T-cell memory generation

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

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

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