Modular Immune Organoids with Integrin Ligand Specificity

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Modular Immune Organoids with Integrin Ligand Specificity Differentially Regulate Ex vivo B Cell Activation Alberto Purwada, Shivem B. Shah, Wendy Beguelin, Ari M Melnick, and Ankur Singh ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00474 • Publication Date (Web): 13 Dec 2016 Downloaded from http://pubs.acs.org on December 13, 2016

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ACS Biomaterials Science & Engineering

Modular Immune Organoids with Integrin Ligand Specificity Differentially Regulate Ex vivo B Cell Activation Alberto Purwada*a, Shivem B. Shah*a, Wendy Beguelinb, Ari M. Melnickb, and Ankur Singhc€ a

Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA

b

Division of Hematology/Oncology, Department of Medicine, Weill Cornell Medical College, New York, NY, USA c

Sibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, NY, USA

*These authors contributed equally to the work.

€

Corresponding Author: Prof. Ankur Singh 389 Kimball Hall, Cornell University, Ithaca, NY 14853-7501, USA E-mail: [email protected] Keywords: B cell receptor, ligand, stromal, differentiation, germinal center, antibody

ACS Paragon Plus Environment


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Abstract Germinal centers are dynamic structures within lymphoid tissues, which develop once B cells receive activating signals from surrounding immune cells. Germinal center B cells are small in number, heterogeneous, and prone to rapid apoptosis unless selected by the body to form memory B cells. Despite extensive research in the B cell differentiation process, the role of the lymphoid niche, in particular integrin ligands, in the development of early germinal center-like phenotype remains unclear. Here we report a biomaterials-based modular immune organoid that enables development of early germinal-center phenotype in an integrin ligand-specific manner. We demonstrate the differential role of integrin α4β1- and αvβ3-binding ligands in the induction of GL7+ (GC-like) and GL7- (non-GC-like) phenotype in differentiating B cells while, in the presence of CD40 ligand and interleukin-4. We further demonstrate the role of integrin ligand specificities in clustering of β3 integrin and B cell receptor on the surface of differentiated B cells in 3D organoids as compared to the classic 2D co-cultures. The study demonstrates that biomaterials-based immune organoids represent an ex vivo platform technology, which recapitulates certain aspects of GC biology to understand the process of B cell differentiation and induction of immunological responses. This platform is particularly useful in understanding the role of selective biomolecular signals and the temporal dependency of immune responses to these signals.

Abbreviation GC MFI BCR PEG PEGMAL DTT VPM RGD REDV CD40L HEPES CPM SRBC

Description Germinal center Median fluorescent intensity B cell receptor Polyethylene glycol PEG with maleimide functionalization Non-degradable crosslinker (also known as “Dithiothreitol”) Degradable crosslinker containing VPM sequence (GCRDVPMSMRGGDRCG) RGD peptide (GRGDSPC) REDV peptide (GREDVGC) CD40 ligand 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Counts per million Sheep red blood cell


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Introduction B cell humoral immunity depends on the germinal center (GC) differentiation process in the B cell follicles of spleen and lymph nodes. GCs are the main sites where antigen‐activated B cell clones expand and undergo affinity maturation1-8. Prior to GC formation, B lymphocytes representing countless specificities are formed through random developmental mutations and stochastic rearrangements of B cell receptor gene segments. During the GC process, naïve B cells first differentiate into GL7+Fas+ phenotype and then undergo additional gene rearrangement to establish high-affinity B cell receptors. Despite extensive research in this area, the underlying mechanisms that modulate the induction of GL7+Fas+ phenotype in B cells and progression of GC reaction remain poorly understood. 4, 5, 9. This lack of understanding can be attributed to the inherent rarity, transience, and heterogeneity of GC B cells. Understanding the mechanisms that favor GC reactions ex vivo may provide better understanding of B cell biology. In the long term, this understanding may also lead to effective strategies for generation of memory B cell responses against a wide range of maladies including cancer, asthma, arthritis, and transplant rejection, along with faster responses to emerging infections such as H1N1 and Zika. Thus far, many primary discoveries in B lymphocytes and GC B cells have focused on clonal heterogeneity and affinity maturation2, 4, 7, 8, 10-12. Most of these discoveries were made using sophisticated mouse models, yet several complementary mechanisms that regulate the initiation and progression of GC reactions remain unclear. In particular, the role of the lymphoid microenvironment, especially the bio-adhesive ligands (i.e. integrin ligands), in modulating the extent of GC reaction and antibody production through the GC pathway is unexplained. Recent studies have elucidated the role of integrin subunits in controlling the B cell phenotype and modulating intermediate context-dependent steps in humoral immunity. Within lymphoid tissues, integrin ligands, such as α4β1-binding vascular cell adhesion protein 1 (VCAM-1), are upregulated on the surface of follicular dendritic cells during GC response to immune complexes13-15. This process is initiated by B cell receptor (BCR) signals and other proteins that lead to inside-out activation of integrins, and subsequent increased integrin avidity for their respective ligands15. In addition, the α4β1 integrin (often called Very Late Antigen-4 (VLA-4)) has been implicated in increasing the adhesion between pre-activated B cells and the ligand-presenting membrane 16,. These adhesions facilitate B cell activation by low-affinity antigen, as indicated by calcium response 16, and ensuring participation inside the GC reaction13. Within the lymphoid microenvironment, integrin αvβ3-binding Arg-Gly-Asp (RGD) motif is also presented by vitronectin within GCs13, 17 and by laminin a5 within the marginal zone of B cell follicles18. While the role of both αv and β3 integrin subunits have been relatively unexplored, they are expressed more in activated B cells 19. β3-definciency has been associated with slight overrepresentation of β3-deficient B cells in the spleen’s GC compartment than its follicular compartment 13 as well as increased sensitivity in marginal zone and peritoneal B-1 B cells towards Toll like receptor (TLR) stimulation19. Furthermore, RGD has been shown to mediate the interaction between B cells and laminin 18. These studies suggest that distinct integrin–ligand interactions can have different influences on the GC response. However, the α4β1 and αvβ3 integrin receptors on B cells can also interact with other integrin ligands in the lymphoid niche, further adding to the complexity of dissecting individual integrin–ligand contributions to the induction of the GC process. In addressing the role of distinct integrin-ligand interactions on the GC responses, current in vivo models can suffer from complexity, inaccessibility, and most importantly, a lack of control over ligand density and selective presentation. Both of these factors may have implications in integrin-mediated feedback signaling, as discussed by Wang et al 13. Additionally, integrin-knockout models can experience altered development 19 or be embryonically lethal 20.



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Although activation of B cells can be achieved ex vivo in simpler and more controllable twodimensional (2D) cultures through stimulation with antibodies (anti-Ig or anti-CD40), CD40 ligand (CD40L), or cytokines such as interleukin (IL)-4, the resulting cell growth is transient, with poor cell yield and rapid apoptosis. 2D co-cultures also exclude 3D presentation of bioadhesive molecules. Both primary limitations of imprecise control and bio-adhesivity could be addressed by 3D biomaterial-based niches, however, biomaterials innovation in B lymphocyte and GCs have been relatively rare in comparison to T cell development 21 and immunotherapy 22 , as reviewed by us elsewhere 23, 24. Current 3D scaffolds have only shown GC-like formation when implanted in vivo by exploiting the host microenvironment 23-26. Importantly, these studies did not provide evidence of control over the rate of GC reaction ex vivo or in vivo, with improved survival of GC-like phenotype. To bridge the functional gap between in vivo and in vitro systems, we have recently developed a gelatin-nanoparticle-based platform to engineer GC-like B cells ex vivo by integrating known structural and signaling components of the lymphoid microenvironment to recapitulate key functional events prior to GC formation 27. In these studies, we demonstrated that 3D immune organoids enable rapid generation of GC-like B cells, with 100-fold generation of CD19+GL7+ GC-like B cells within 4 days of co-culture in 3D organoids as compared to 10-fold generation of the same phenotype in a classic 2D co-culture. However, gelatin presents abundant RGD ligand, which makes it difficult to study the role of specific integrin ligands, including α4β1-binding ligands, in the development of GC-like phenotype. Here, we present a modular B cell follicle organoid using thiol-crosslinked maleimide polyethylene glycol (PEGMAL)28 which presents specific integrin ligands at controlled density to B cells, which are co-cultured with CD40L-presenting stromal cells. This approach allows us to investigate the role of specific integrin ligand interactions during the T cell dependent B cell activation process (without the need for antigen) with regards to B cell phenotype development. 1. Materials and methods 2.1 Polymers, peptides, and compounds: Four-arm maleimide-functionalized polyethylene glycol (PEGMAL) with 20 kDa molecular weight was purchased from Laysan Bio, Inc. (Arab, AL) with above 90% purity. Integrin αvβ3-binding RGD peptide29-31 (GRGDSPC, 96% purity), integrin α4β1-binding REDV peptide32 (GREDVGC, 96% purity), and matrix metalloproteinase 9 degradable VPM peptide (GCRDVPMSMRGGDRCG) were purchased from AAPPTec, LLC (Louisville, KY) with above 90% purity. Integrin αvβ3 inhibitor Cilengitide (a cyclic RGD) was purchased from Selleck Chemicals. Mitomycin C was purchased from Santa Cruz Biotechnology. Murine recombinant IL-4 was purchased from Peprotech (Rocky Hill, NJ). 2.2 In vivo immunization to characterize GC B cells: Two C57BL6 mice were immunized intraperitoneally at 8 weeks of age with 0.5 ml of a 2% sheep red blood cell (SRBC) suspension in PBS (Cocalico Biologicals), and sacrificed after 8 days. Single-cell suspensions from mouse spleens were stained using the following fluorescent-labeled anti-mouse antibodies: PerCPCy5.5 conjugated anti-B220, PECy7 conjugated anti-FAS, AlexaFluor 647 conjugated anti-GL7 (BD Biosciences), PE conjugated anti-integrin α4, PE conjugated anti-integrin αV, FITC conjugated anti-integrin β1, FITC conjugated anti-integrin β3, PE conjugated IgG2bκ isotype control, PE conjugated IgG1κ isotype control, and FITC conjugated IgG isotype control (eBioscience). DAPI was used for the exclusion of dead cells. GC B cells were identified as B220+ FAS+ GL7+, and NB cells as B220+ FAS- GL7-. Levels of integrin expression versus isotype control were determined within NB and GC B cell populations. Data were acquired on BD FACSCanto flow cytometer (BD Biosciences) and analyzed using FlowJo software package (TreeStar). 2.3 Primary naïve B cells and CD40L-presenting stromal cells: Spleens were harvested from female C57BL/6 mice that were aged 10-16 weeks and purchased from the Jackson Laboratory


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(Bar Harbor, ME). Naïve B cells were isolated from digested spleen via negative selection through the CD19 EasySep Mouse B Cell Isolation Kit, purchased from Stem Cell Technologies (Vancouver, Canada). CD40L-presenting stromal cells were generated from NIH/3T3 fibroblasts genetically engineered to express membrane-bound CD40L and secrete B cell activating factor (BAFF), as previously described by us 27 and Nojima et al 33. This cell line, called 40LB hereafter, was cultured with high glucose DMEM media containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S); all components were bought from Thermo Fisher Scientific (Waltham, MA). All procedures were approved by Cornell University's Institutional Animal Care and Use Committee. 2.4 Antibodies: Antibodies used for flow cytometry included anti-mouse CD19 (eBioscience, PeCy7, eBio1D3), anti-mouse IgM (eBioscience, FITC, eB121-15F9), anti-mouse IgG1 (eBioscience, APC, M1-14D12), anti-mouse integrin αv (eBioscience, PE, RMV-7), anti-mouse integrin β3 (eBioscience, FITC, 2C9.G3), anti-mouse α4 (eBioscience, PE, R1-2), anti-mouse β1 (eBioscience, FITC, eBioHMb1-1), anti-mouse GL7 (eBioscience, FITC and APC, GL-7), and anti-mouse Fas (BD, APC, Jo2). Antibodies used for confocal microscopy included Armenian hamster anti-mouse integrin β3 (eBioscience, purified, 2C9.G3), rat anti-mouse CD19 (eBioscience, purified, eBio1D3), rabbit anti-mouse IgM (Abcam, purified, II/41), goat antiArmenian hamster (Abcam, Alexa Fluor 488 secondary antibody), goat anti-rat (Abcam, Alexa Fluor 594 secondary antibody), and goat anti-rabbit (Thermo Fisher Scientific, Alexa Fluor 633 secondary antibody). For antibody blocking studies, anti-mouse α4 (clone 9C10 (MFR4.B)) and anti-mouse β1 (clone HMβ1-1) were purchased from Biologend and used at 2 µg/mL concentration. 2.5 PEGMAL organoid fabrication process: Synthetic hydrogel organoids with 7.5% (w/v) macromer concentration were fabricated using PEGMAL (Laysan bio, Inc., >90% purity) and thiolated crosslinkers. PEGMAL macromers were initially functionalized with thiolated bioadhesive peptides RGD or REDV with 4:1 macromer-to-peptide molar ratio. MMPdegradable peptide and non-degradable dithiothreitol (DTT) thiolated crosslinkers were combined at a 50:50 molar ratio and 4:1.5 macromer-to-crosslinker molar ratio. All components were diluted using PBS++ solution with pH 7.4 and 1% HEPES. Naïve B cells with 40LB stromal cells were prepared and suspended in the crosslinker solution prior to cell encapsulation. After 5 µL of PEGMAL macromer was placed in the middle of a well of a non-treated 96 well plate, 5 µL of cell-containing crosslinker solution was injected into the initial droplet and mixed by pipetting up and down 5 times. Hydrogel droplets were prepared and cured for 15 min at 37° C inside cell culture incubator for complete crosslinking. Fresh RPMI 1640 media supplemented with 10% FBS, 1% P/S, and 10 ng/mL IL-4 was then added to the organoid culture. 2.6 Confocal microscopy imaging: In order to observe the morphology and the clustering of surface markers, synthetic hydrogel organoids were prepared as described above using uncoated glass-bottom (no. 1.5 coverslip and 20 mm glass diameter) 35mm dish, purchased from MatTek Corporation. All organoids were functionalized with 3 mM integrin ligand density. On day 4 post-encapsulation, the organoids were fixed with 4% paraformaldehyde, rinsed with PBS++, incubated with blocking buffer, stained with primary antibodies overnight, then stained with secondary antibodies for 4 h afterwards, and immediately imaged using Zeiss LSM 710 confocal microscope. Blocking buffer was prepared using PBS++ containing 20% goat serum. Staining was performed using flow buffer with antibodies diluted using antibody-to-buffer volume ratio of 1:500. Washing was done using PBS solution. 2.7 Flow cytometry: After 4 days of culture, cells were harvested from the hydrogel organoids by enzymatic degradation for 1 hr using a 125 U/mL collagenase-1 from Worthington Biochemical



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(Lakewood, NJ), dissolved in serum-free RPMI medium. Cells were then collected by passing the cell suspension through a 70 µm cell strainer (BD Falcon). Cells were re-suspended in flow buffer containing antibodies (1:500 dilution) and washed two times with the flow buffer solution. Flow buffer solution was prepared using PBS++ containing 2% (v/v) FBS and 5 mM EDTA. The cell suspension was analyzed using Accuri C6 flow cytometer from BD Biosciences (San Jose, CA). 2.8 Statistical analysis process: Analysis of variance (ANOVA) statistical analyses was performed using GraphPad Prism software with Tukey's test (one-way ANOVA). A p-value of less than 0.05 was considered significant. All studies were performed with five technical triplicates unless otherwise noted. All experiments were repeated multiple times. All values are reported as mean ± SEM. 2. Result and Discussion 3.1 GC B cells differentially express integrin α4β1 and αvβ3 genes In B cell studies, the focus of integrin expression and clustering analysis has been inside-out signaling, mediated by B cell receptors (BCRs) that capture antigens 34-36. Although integrin mediated outside-in signaling has been implicated in T cells, its role in the initiation of early GC-like B cell differentiation process remains unclear. Importantly, the roles of selective integrin ligand presentation on differentiation of naïve B cells into GL7+Fas+ GC-like phenotype and BCR fate remain unclear. We first examined a previously published genome-wide gene expression analysis from the National Institute of Health (NIH) repository to understand the integrin gene expression levels in naïve B cells versus GC-like B cells in mice. Selective reanalysis of a study by Dominguez et al. 37 indicated that the expression levels of integrin subunits α2, α5, and α6 were very low for both naïve B cells and differentiated GC B cells (Figure 1A). The α4 integrin subunit on the other hand was highly expressed in total naïve B cells (317 counts per million (CPM) and reduced to 67 CPM upon differentiation into GC phenotype). β1 integrin subunits had a lower expression level in naïve B cells with 36 CPM, which increased to 81 CPM in GC B cells. We next evaluated the expression levels of αv and β3 integrins and observed 22-24 CPM for αv and 48-50 CPM for β3 integrin subunits in both naïve and GC B cells. These studies indicate that α4 and β1 markedly change during GC process, whereas αv and β3 integrins are expressed constantly at the same levels. We next determined the surface expression of integrins α4, αv, β1, and β3 in in vivo immunized C57BL6 mice. Sheep red blood cell (SRBC) immunized mice developed GC over 8 days and splenocytes were co-stained with B220+ and hallmark GC surface marker GL7 and Fas (Figure 1B). Analysis of integrin subtypes on B200+GL7+Fas+ cells indicated that percentage of integrins αv, β1, and β3 expressing cells were significantly higher in differentiated GC B cells as compared to naïve B cells (Figure 1C), whereas percentage of integrin α4 expressing cells were comparable for naïve B cells and GC B cells. We also evaluated the expression levels of these integrins between naïve and GC B cells and observed similar pattern as observed with percent cells expressing these surface receptors (Figure 1D). We used this information to understand the role of these subunits in GC-like phenotype and whether the induction of GL7+ phenotype changes with alterations in integrin-binding ligand density or co-presence of multiple integrin ligands. We engineered a modular B cell follicle organoid using a 4-arm thiol-crosslinked maleimide polyethylene glycol (PEGMAL) hydrogel, which we have previously reported to culture malignant B cells28. Physical characterization of this hydrogels have been previously reported by Phelps et al38 and us28. Briefly, one or more arms of the PEGMAL can be functionalized with thiolated adhesive peptide and the resulting functionalized PEG macromers are subsequently cross-linked into a hydrogel by addition of either non-degradable crosslinker


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dithiothreitol or a dithiol protease-cleavable peptide cross-linker (Figure 2A) to allow for stromal cell spreading. It has been previously reported that RGD concentrations ranging between 0.025 mM and 3.5 mM support 3D cell adhesion and spreading in PEG-based hydrogels 38. We designed our studies to functionalize one arm of the four-arm PEGMAL polymer with thiolated adhesive peptides and therefore used an adhesive peptide concentration of 3.0 mM to maximize adhesion sites while retaining cross-linking ability in 4-arm PEGMAL. If more than 1arm of the 4-arm PEGMAL was occupied per PEGMAL precursor, it is likely that a crosslink may not form or a non-crosslinking cyclization may occur as discussed earlier by Metters and Hubbell 39. Although we did not quantify adhesive peptide incorporation, previous study by Phelps et al38 has shown 100% incorporation of thiolated RGD peptides at MAL:RGD molar ratios 1:1 and higher. A key cellular component in the immune organoids is the CD40L-presenting stromal cells that secrete BAFF. Stably transduced BALB/c 3T3 fibroblasts with CD40L and BAFF were used (cells are called 40LB33), which effectively substituted for Follicular T helper cells (Tfh) cells and follicular dendritic cells, key immune cell types involved in B cell activation 33. In other words, the 40LB system provided the CD40L signal that would normally come from Tfh cells to emulate “T cell” dependent manner responses 40, 41. Naïve B cells with 40LB cells were resuspended in the polymer mixture and co-encapsulated in the resulting organoids. The method of preparation of organoids is highly reproducible however slight experimental variation in exact droplet geometry of organoids was observed during the pipetting process. For the induction of GC-like phenotype, we have previously shown that spreading of 40LB stromal support was critical 27, 42. Several previous studies have shown that hydrogels with protease degradable crosslinkers support cell spreading and survival 38, 43-47. In particular both 3T3 fibroblasts48 as well as B cells49 are known to secrete matrix metalloprotease-9. In our studies, we examined the effect of matrix degradability on the spreading of CD40L-presenting stromal cells (called 40LB) and observed that hydrogels with non-degradable crosslinker DTT resulted in aggregated cells and did not support spreading of 40LBs (Figure 2B). In contrast, when PEGMAL was crosslinked with matrix metalloprotease 9-degaradable GCRDVPMSMRGGDRCG peptides (mixed with DTT at 1:1), superior cell spreading was observed. Prior to attempting the 1:1 ratio of VPM and DTT, we also tested the degradability of hydrogels fabricated using a 100% VPM cross-linker and observed fast degradation times due to 40LB cells (data not shown), thereby precluding the use of only VPM. We therefore used VPM:DTT (1:1) hydrogels and then functionalized them with a range of RGD peptide ligand density (0.003 – 3 mM) to determine the effect of adhesive ligand density on 40LB spreading. As indicated in Figure 2C, at lower ligand densities of 0.003 – 0.3 mM, 40LB cells cluster or do not form a spread cell network, whereas at 3 mM ligand density, 40LBs demonstrate spread network. Therefore for the remainder of the studies we examined the effect of RGD (1.5 and 3.0 mM), REDV (1.5 and 3.0 mM) and a combination of RGD and REDV (1.5 mM each). We first characterized the role of REDV versus RGD ligand presentation on 40LB spreading and CD40L expression. As indicated in Figure 2D, 40LBs spread uniformly with wellformed actin fibers on both REDV- and RGD-functionalized organoids with 3 mM ligand density. In both organoid systems, 40LB cells demonstrated equivalent CD40L expression levels (Figure 2E), with slightly higher Median Fluorescence intensity (MFI) when cultured in RGDtethered organoids. 3.2 Integrin ligand specificity determines the fate of B cell activation We hypothesized that immune organoid-mediated selective integrin ligand presentation will differentially regulate the B cell differentiation into early GC-like phenotype (GL7+) in the presence of 40LB cells. To test this hypothesis, CD19+ naïve B cells, freshly isolated from mice spleen, were co-cultured with 40LB cells in organoids in the presence of IL-4 (10 ng/mL). Prior studies have shown that IL‐4 exchanged during early cognate interaction between B and T cells



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is important for the induction of GC B cell differentiation 50. After 4 days of differentiation, we did not observe any significant differences between number of proliferated CD19+ B cells across tested ligand types and densities (Figure 3A, B). Since activated and mature GC B cells express the GL7 epitope 51-54; we examined GL7 as a marker for early GC-like phenotype. Analysis of CD19+GL7+ phenotype revealed that at 1.5 mM and 3 mM ligand density, REDV-functionalized organoids resulted in significantly higher percentage of CD19+GL7+ cells as compared to RGD-functionalized organoids (Figure 3C, p < 0.05). Within REDV or RGD-containing organoids, a 2-fold change in ligand density did not result in any significant change in CD19+GL7+ phenotype. To further evaluate changes in the GC phenotype, we studied CD95 (Fas/APO-1), a highly characterized member of the proapoptotic receptor family, as the strongest expression of CD95 in the mature B cell compartment is found on GC B cells 55. Fas-mediated selection of GC B cells and the resulting memory B cell compartment is essential for maintaining the homeostasis of both T and B lymphocytes and GC B cells are susceptible to Fas-mediated apoptosis in vitro 56. After evaluating the CD19+GL7+Fas+ cells, we observed no differences between the phenotype when differentiated in the presence of 1.5 mM REDV or RGD ligands. However, increasing the REDV peptide ligand density to 3 mM resulted in a significant increase in CD19+GL7+Fas+ phenotype (Figure 3D, p < 0.05). A similar two-fold increase in RGD ligand density to 3 mM did not result in any significant change in the total percentage of GC-like B cells. Since multiple ligands are simultaneously present in a lymphoid niche, we next determined the effect of 50:50 presentation of REDV+RGD ligands (each at 1.5 mM) and observed same percentage of cell expressing CD19+GL7+Fas+ phenotype as observed with 1.5 mM RGD-functionalized or REDV-functionalized organoids. Based on these observations and our previously published RGD-organoid work,27 we postulated that although RGD-ligands drive the GC-like reaction better than 2D conditions, α4β1-binding ligands enhance GC differentiation. Because simultaneous presentation of REDV and RGD ligand did not enhance GC-like differentiation more than RGD alone, we concluded that αvβ3 possibly has a dominating effect when presented simultaneously in a niche. This conclusion is supported by prior studies where integrin β3-deficienct B cells contributed in a slightly exaggerated manner to GC responses, highlighting a regulatory role of integrin β3 in GC B cells 13. To validate B cell activation is driven by CD40L and not biomaterial immunogenicity, we cultured B cells in RGD functionalized PEGMAL organoids with and without 40LB cells. As indicated in Figure 3E, PEGMAL hydrogels without 40LB cells failed to induce B cell proliferation and activation, whereas those seeded with 40,000 40LBs per 10 µL organoid resulted in significantly higher GC-like B cells. We then determined the temporal dependency of GL7+ phenotype emergence on αvβ3 integrin-ligand interactions by using a cyclic RGD pentapeptide drug Cilengitide, a potent integrin inhibitor for αvβ3 receptor and αvβ5 receptor that is currently in clinical phase III for treatment of glioblastomas and in phase II for several other tumors 57. As indicated in Figure 4A, a significant reduction in the percentage of CD19+GL7+ B cells, an early indicator phenotype of GC-like B cells, was observed only when the αvβ3 integrin-ligand interaction was blocked at t = 0. Addition of Cilengitide at other time points (1, 4, 12 h) resulted in no change in percentage of GC-like B cells as compared to untreated control group, indicating that early integrin-ligand interactions are critical for generation of GC-like phenotype. Although similar inhibitory effects were observed in CD19+GL7- B cells, overall proliferation of this non-GC-like phenotype was significantly higher than GC-like phenotype in RGD-organoids when Cilengitide was added after 1 h. We next determined the temporal dependency of B cell activation on outside-in integrin signaling by selectively inhibiting integrins α4 and β1 interactions with REDV peptide ligand using antibodies against α4 or β1. As indicated in Figure 4B, inhibition of α4 integrin at t = 0 did not inhibit GL7+ phenotype, however β1 inhibition at t = 0 inhibited induction of GL7+ phenotype. Inhibition of α4 or β1 at later time points did not inhibit differentiation of


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naïve B cells into GL7+ phenotype. These observations indicate that for GC-like phenotype, early integrin-ligand interaction is critical. Our observations on integrin α4 inhibition at t = 0 are further supported by in vivo studies reported earlier by Wang et al 58, which had suggested that α4 integrin function on B cells was not crucial for entry into the GC response. The decrease in B cell proliferation and GC induction could also be attributed to possible inhibitory effect of Cilengitide on 40LB stromal cells adhesion to peptide ligands in hydrogels. Because 40LB spreading is important for induction of GC-like phenotype 27, we evaluated the effect of Cilengitide on REDV-functionalized organoids in which 40LB cells are normally spread (Figure 2D). We observed no inhibition of GC-like phenotype (Figure 4A). Importantly, we observed an increase in GL7+ cells in REDV-functionalized organoids when αvβ3 was blocked, suggesting that αvβ3 was indeed expressed under non-RGD growth conditions and possibly has a regulatory role in the induction of GC-like B cell. To confirm whether integrin β3 is expressed even in REDV-functionalized organoids, we performed confocal imaging (Figure 5) and observed that integrin β3 clustered under both RGD and REDV organoids, which suggest that integrin β3 is central to the ex vivo GC phenotype development. No such clustering was observed in 2D co-culture conditions (Figure 5), suggesting the need for a 3D niche to study ex vivo B cell differentiation and induction of GC-like phenotype. These observations further suggest that adhesive-ligands possibly drive the ex vivo GC-like reaction better than 2D conditions through integrin clustering. 3.3 Integrin ligand composition differentially regulate the integrin expression on GC-like and non-GC-like B cells We next quantified the expression level of integrin α4, β1, αv, and β3 on the surface of CD19+ B cells on day 4, the peak time for GC-like differentiation in organoids 27. As indicated in Figure 6A, integrin α4 expression level (MFI) was significantly higher in REDV-functionalized organoids than RGD-functionalized organoids, at both 1.5 and 3 mM ligand density. A two-fold increase in ligand density to 3 mM did not result in any significant change in integrin α4 expression level. We next determined the effect of 50:50 presentation of REDV+RGD ligands (each at 1.5 mM) and observed expression levels of integrin α4 similar to those found in 1.5 mM RGD-functionalized organoids and significantly lower than those in 1.5 mM REDV-functionalized organoids. Our studies demonstrate that while α4β1-binding REDV ligand leads to higher expression of integrin α4 than those in RGD ligand, simultaneous presentation of REDV and RGD ligands has a dominating effect of RGD over REDV. This situation could be comparable to in vivo conditions where both ligands are simultaneously present in GC,as is the case for secreted vitronectin within GCs13, 17. Unlike the integrin α4 expression levels, the fluorescence intensity of integrin β1 staining had peaks that represent high and low β1 surface expression levels (Figure 6B, C). Expression level of integrin β1lo in CD19+GL7+ cells followed similar pattern as integrin α4, where MFI values in REDV-presenting organoids were significantly higher than RGD presenting immune organoids (p

Modular Immune Organoids with Integrin Ligand Specificity

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