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B Cells Loaded with Synthetic Particulate Antigens: A Versatile Platform to Generate Antigen-Specific Helper T Cells for Cell Therapy Antoine Sicard, Alice Koenig, Stéphanie Graff-Dubois, Sébastien Dussurgey, Angéline Rouers, Valérie Dubois, Pascal Blanc, Dimitri Chartoire, Elisabeth Errazuriz-Cerda, Helena Paidassi, Morgan Taillardet, Emmanuel Morelon, Arnaud Moris, Thierry Defrance, and Olivier Thaunat Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.5b03801 • Publication Date (Web): 09 Dec 2015 Downloaded from http://pubs.acs.org on December 14, 2015
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French National Institute of Health and Medical Research (INSERM) Unit 1111; Claude Bernard Lyon 1 University; Ecole Normale Supérieure de Lyon; CNRS, UMR 5308; Edouard Herriot Hospital, Transplantation, Nephrology and Clinical Immunology Department Moris, Arnaud; Sorbonne University, UPMC Univ Paris 06, INSERM U1135, CNRS ERL 8255, Center for Immunology and Microbial Infections – CIMIParis, F-75013 Defrance, Thierry; International Center for Infectiology Research (CIRI); French National Institute of Health and Medical Research (INSERM) Unit 1111; Claude Bernard Lyon 1 University; Ecole Normale Supérieure de Lyon; CNRS, UMR 5308 Thaunat, Olivier; International Center for Infectiology Research (CIRI); French National Institute of Health and Medical Research (INSERM) Unit 1111; Claude Bernard Lyon 1 University; Ecole Normale Supérieure de Lyon; CNRS, UMR 5308; Edouard Herriot Hospital, Transplantation, Nephrology and Clinical Immunology Department
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B Cells Loaded with Synthetic Particulate Antigens: A Versatile Platform to Generate Antigen-Specific Helper T Cells for Cell Therapy
Antoine Sicard1,2, Alice Koenig1,2, Stéphanie Graff-Dubois3, Sébastien Dussurgey4, Angéline Rouers3, Valérie Dubois5, Pascal Blanc1, Dimitri Chartoire1, Elisabeth ErrazurizCerda6, Helena Paidassi1, Morgan Taillardet1, Emmanuel Morelon1,2, Arnaud Moris3, Thierry Defrance1, Olivier Thaunat* 1,2
1) International Center for Infectiology Research (CIRI); French National Institute of Health and Medical Research (INSERM) Unit 1111; Claude Bernard Lyon 1 University; Ecole Normale Supérieure de Lyon; CNRS, UMR 5308, Lyon, France; 2) Edouard Herriot Hospital, Transplantation, Nephrology and Clinical Immunology Department, Lyon, France; 3) Sorbonne University, UPMC Univ Paris 06, INSERM U1135, CNRS ERL 8255, Center for Immunology and Microbial Infections – CIMI-Paris, F-75013, Paris, France 4) SFR Biosciences, UMS344/US8, Inserm, CNRS, Claude Bernard Lyon-1 University, Ecole Normale Supérieure, Lyon, France. 5) French National Blood Service (EFS), Lyon, France; 6) Center for Quantitative imaging (CIQLE), SFR Santé Lyon-Est, Lyon 1 University, Lyon, France
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ABSTRACT Adoptive cell therapy represents a promising approach for several chronic diseases. This study describes an innovative strategy for biofunctionalization of nanoparticles, allowing the generation of synthetic particulate antigens (SPAg). SPAg activate polyclonal B cells and vectorize non-cognate proteins into their endosomes, generating highly efficient stimulators for ex vivo expansion of antigen-specific CD4+ T cells. This method also allows harnessing the ability of B cells to polarize CD4+ T cells into effectors or regulators.
KEYWORDS: bioengineered nanoparticle, biomimetic, B cell, antigen-presenting cell, CD4+ T cell, cell therapy
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TEXT
For decades, immunotherapy has exclusively relied on in vivo administration of pharmacological preparations aiming at either stimulating (i.e. vaccines) or dampening (i.e. immunosuppressive drugs) patients’ immune responses. Based on promising results obtained in animals models, the concept of ex vivo manipulation of immune cells for retransfer as cell therapy: “adoptive cell therapy” (ACT), has progressively emerged1,2. The demonstration in the late 80’s that the transfer of ex vivo expanded tumor-infiltrating T lymphocytes to patients with melanoma could lead to cancer regression paved the way for the translation of ACT to the clinic3. ACT is currently considered as a central strategy to treat severe chronic conditions as diverse as viral infections, cancers, autoimmune diseases, allograft rejection or graft versus host disease (218 clinical studies currently registered in https://clinicaltrials.gov/). Much of initial experimental and clinical studies in ACT have focused on cytotoxic CD8+ T cells because of their remarkable ability to kill tumors or virus-infected cells2,4-7. However, attention has progressively shifted to helper CD4+ T cells, which are endowed with a much wider spectrum of functions2,8,9. Indeed, CD4+ T cells are not only very efficient for tumor and virus destruction through direct cytotoxicity and promotion of cytotoxic CD8+ T cells responses10-14 but also necessary for the generation of protective antibody-responses15. Besides the promotion of effector responses, CD4+ T cells are also endowed with unique immune regulatory properties16. The versatility of CD4+ T cells is due to their plasticity, which allows them to differentiate into various functional subsets according to the microenvironment in which they are activated16. Importantly, several experimental studies have illustrated that ACT was more efficient and associated with fewer side effects when antigen-specific CD4+ T cells were used instead of polyclonal CD4+ T cells2,8,9,17. Two recent studies have validated this concept in the clinic.
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First, ex vivo-expanded autologous CD4+ T-cell clones specific for a given melanomaassociated antigen were able to induce durable clinical remission in a patient with refractory metastatic melanoma18. Second, adoptive transfer of CD4+ T cells recognizing a unique tumor epitope could mediate regression of a metastatic epithelial cancer19. Such antigenspecific CD4+ T cells are however rare and need to be specifically expanded for ACT20. Nanobiotechnology represents a powerful tool to reach this crucial objective 21-28. Physiologically, clonal expansion of antigen-specific CD4+ T cells requires engagement of the T cell antigen-specific receptor (TCR) by the antigen-class II major histocompatibility complex (MHC-II) on the surface of antigen-presenting cells (APCs). Although reports have shown that it was possible to expand antigen-specific T cells ex vivo with artificial antigen presentation by cell-free substitutes, these emerging technologies still warrant preclinical and clinical validations21,29. Current clinical approaches aiming at expanding CD4+ T cells clones for ACT rather rely on autologous APCs that can be reliably used to confer optimal therapeutic features to T cells before infusion18,19. Dendritic cells (DCs) are professional APCs that can be readily pulsed with any antigen and used as stimulators of antigen-specific CD4+ T cells30. Yet, implementation in practice is difficult because DCs are too rare to be directly purified from peripheral blood. Instead, bone marrow or blood progenitors must be matured in culture during several days before being used as T cell activators. This procedure increases the costs and leads to inconstant yields31-34. Furthermore, the number of DCs that can be obtained from these cultures is limited as mature DCs stop dividing and become less effective at presenting antigen after 2 to 3 weeks in culture (in fact, it is generally accepted that the final number of DCs cannot be expanded beyond the number of starting progenitors)33,34. In contrast, B cells are “ready to use” APCs, which are abundant in the circulation (up to 0.5x106 cells/mL) and can be further exponentially expanded in vitro without loss of antigen-presenting functions31-33. B cells
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therefore represent an unrestricted source of autologous APCs for ACT31,32. However, the use of B cells as stimulators of antigen-specific CD4+ T cells is made problematic due to their inability to present non-cognate antigens35. In contrast with DCs, which can engulf any antigen by phagocytosis, B cells can only internalize a cognate antigen36-38. It is indeed the binding of the specific antigen to B cell’s surface immunoglobulins (B cell receptor, BCR) that triggers: (i) the activation signal required for the acquisition of potent antigen-presenting functions and (ii) the antigen internalization in endosomes where antigen is processed and loaded onto MHC-II for presentation to CD4+ T cells. Antigen-specific B cells are too rare to be used in ACT. To overcome this obstacle, we have developed an innovative strategy for biofunctionalization of nanoparticles. Bioengineered nanospheres were specifically designed to simultaneously: i) provide BCR-dependent activation signal, and ii) deliver non-cognate antigens to endosomes of B cells. Using these Synthetic Particulate Antigens (SPAg), we were able to turn resting polyclonal B cells into potent stimulators of antigen-specific CD4+ T cells. To generate SPAg, proteins of interest were biotinylated and immobilized on fluorescent streptavidin-coated polystyrene nanospheres of 400 nm in diameter, a size comparable to that of a typical pathogen. These nanospheres (Bangs laboratories, Indiana, USA) were generated by an emulsion polymerization synthesis using a sulfate-based initiator. To optimize the tracking of nanospheres, the fluorescent dye was embedded within the core of the nanospheres to ensure the protection of fluorescent molecules from proteolysis processes that take place within some cellular compartments. Following synthesis, streptavidin was covalently bound on nanospheres surface and a blocker was applied (for more detail please refer to online supplemental information). The streptavidin/biotin system was chosen to coat proteins of interest on nanospheres because it provides interactions that have similar strength than covalent bonds (Kd≈10-14 mol/L) while remaining simple to manipulate. Another
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advantage of this system is that it allows using spacer arms that, by separating the biotinylated ligand from the surface of the nanospheres, reduce the steric hindrance. Nevertheless, other coupling strategies could have been chosen. For instance, we successfully generated SPAg by coating purified proteins on carboxyl modified nanospheres using carbodiimide as crosslinking reagent but the yields and the quality of the final product was consistently lower (data not shown). BCR is composed of 2 pairs of polypeptides chains: 2 heavy chains and 2 light chains. Light chains can be of two types: lambda or kappa. More than 90% of murine B cells express kappa light chains. Each chain comprises both a constant domain, which is a shared framework independent of the antigenic specificity, and a highly variable domain, which is specific to each B cell clone and involved in the recognition of antigenic epitopes. We reasoned that coating the nanospheres with a biotinylated monoclonal antibody directed against a framework region of kappa light chain (anti-κ mAb) would confer them the capacity to target any non-cognate kappa positive BCR while behaving like genuine particulate antigens (Figure 1a). The coating of nanospheres with anti-κ mAb was verified by electron microscopy (EM) using gold particle-coupled anti-IgG secondary antibody (Figure 1b). To ensure that anti-κ mAb were attached to nanospheres by streptavidin-biotin solid bonds rather than unspecific adsorption, coating efficiency using biotinylated versus purified anti-κ mAb was compared in flow cytometry with a PE-conjugated anti-IgG antibody (Figure 1c). The successful double coating of nanospheres with biotinylated anti-κ mAb and biotinylated ovalbumin, which was used herein as model antigen, was verified by flow cytometry (Figure 1d). To limit flocculation of nanospheres caused by the coating procedure, anti-κ mAb and ovalbumin had to be monobiotinylated rather than polybiotinylated. This was objectified by the analysis of the particles size in flow cytometry (Figure 1e). A final filtration of coated particles allowed to obtain a solution with more than 90% of mono- or bi- dispersed SPAg, as
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shown by the analysis in imaging flow-cytometry of SPAg fluorescence before and after filtration (Figure 1f). An estimation of the mean number of proteins that can be coated on each nanosphere was obtained by incubating nanospheres with increasing amounts of monobiotinylated ovalbumin or anti-κ mAb and quantifying the binding by flow cytometry with anti-ovalbumin or anti-IgG mAbs (Figure 1g). Based on the saturation curves obtained using this method, we evaluated that the maximum number of ovalbumin and anti-κ mAb molecules than can be coated on each SPAg was approaching 10.104 and 15.104 respectively (Figure 1g). The final concentration of SPAg after filtration was measured by spectrophotometry using the standard curve method (Figure 1h). To test the hypothesis that SPAg would bind to any non-cognate kappa positive BCR and behave like genuine antigens, 400 nm diameter SPAg were incubated in vitro with B cells purified from the spleen of a wild type mice at a 100:1 SPAg to B cell ratio. Each of the steps necessary for antigen-presentation by B cells were analyzed: (i) attachment to surface BCR, (ii) triggering of activation signal and (iii) internalization in late endosomal compartment37 (Figure 2a). Flow cytometry analysis showed that SPAg bound exclusively to B220+ lambda chain- B cells, confirming that SPAg interaction with B cells was dependent upon anti-κ mAb (Figure 2b). Noteworthy, SPAg were able to bind with equal efficiency to the two main subsets of mature splenic B cells (follicular (FO) and marginal zone (MZ); Figure 2c). Flow cytometry results were confirmed by EM analyses (Figure 2d). To assess whether SPAg were able to trigger BCR signaling cascade, as would cognate Ag, imaging flow cytometry was used to detect the phosphorylated form of the B cell linker protein (p-BLNK), an adaptor protein that is phosphorylated upon BCR crosslinking39 (Figure 2e). A diffuse p-BLNK signal was detected in both lambda and kappa positive B lymphocytes, when the cells were activated with soluble anti-IgM (Fab’)2 (positive control). In contrast, incubation of B lymphocytes with SPAg resulted in a punctiform p-BLNK signal, which colocalized with SPAg
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fluorescence and was only detected in kappa-positive B cells. More than sixty percent of SPAg bound to B cell surface had triggered BCR signaling 3 minutes post-incubation (Figure 2e, right). It has been demonstrated that because multivalent antigens are able to bring several BCRs complex in close apposition, they are far more potent B cells stimulators than monovalent antigens37,40. We reasoned that nanospheres might allow exploiting this specificity of BCR-mediated B cell activation (Supplemental Figure 1a). Consistent with this hypothesis, a similar level of p-BLNK was obtained with ∼4 times less anti-κ mAbs when using nanoparticles (Supplemental Figure 1b). By reducing the number of anti-κ mAb molecules required to trigger BCR signaling, a nanosphere platform allows maximizing the amount of antigen vectorized in B cells. This is important since we have shown previously that the ability of B cells to activate antigen-specific CD4+ T cells directly correlates with the amount of internalized antigen41. The internalization of SPAg, which is compulsory for antigen presentation, was analyzed by EM and quantified overtime with imaging flow cytometry. Nanospheres with diameters of 3 µm, 400 nm or 110 nm were coated using the same solutions of monobiotinylated anti-κ mAb and ovalbumin and compared. Attachment to B cell surface was observed with the 3 sizes of SPAg (Figure 2d and Supplemental Figure 2a) but only 110 and 400 nm SPAg were internalized by B cells (Figure 2f and Supplemental Figure 2b). After fifteen hours in culture, 98% of 400 nm SPAg had been internalized in B cells (Figure 2g). A confocal microscopy analysis revealed the co-localization of 400 nm SPAg with a marker of late endosomes (Lamp1) and showed that internalized SPAg were situated in the late endosomal compartment, where antigens are processed and loaded onto MHC-II (Figure 2h). In ordinary conditions, the subsequent migration of antigen-MHC-II complexes at the surface of B cells and the expression of costimulation molecules (CD80/86) lead to the activation and proliferation of cognate CD4+ T cell clones (Figure 3a). Consistently, flow
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cytometry analyses after overnight culture showed a higher expression of MHC-II and CD86 in B cells that had internalized 400 nm SPAg, as compared with controls (Figure 3b, 46113±6888 vs 12504±2577 and 3084±505 vs 730±169 respectively, p
