Antibody-secreting cells are generated in regional lymphoid tissues and traffic as plasmablasts (PBs) via lymph and blood to target sites for local immunity. We used multiparameter flow cytometry to ...define PB trafficking programs (TPs, combinations of adhesion molecules and chemoattractant receptors) and their imprinting in patients in response to localized infection or immune insults. TPs enriched after infection or autoimmune inflammation of mucosae correlate with sites of immune response or symptoms, with different TPs imprinted during small intestinal, colon, throat, and upper respiratory immune challenge. PBs induced after intramuscular or intradermal influenza vaccination, including flu-specific antibody-secreting cells, display TPs characterized by the lack of mucosal homing receptors. PBs of healthy donors display diverse mucosa-associated TPs, consistent with homeostatic immune activity. Identification of TP signatures of PBs may facilitate noninvasive monitoring of organ-specific immune responses.
Abstract
Flow cytometry is an essential technology to identify and quantify cell populations. Here we examined the development of new fluorochromes that could enhance flow cytometry’s capability. The ...latest spectral flow cytometers have 3 excitation lasers (405nm, 488nm, and 640nm) and incorporate the Avalanche Diodes Photodetector technology, which demonstrates significant improvement in sensitivity for the fluorescent emission signal longer than 800nm. However, there is no commercially available fluorochrome which could be excited by the above lasers and has peak emission signal beyond 800nm. To address the gap in technology, here we engineered 6 new fluorochromes: PE-750, PE-810, PE-830 for blue laser and APC-750, APC-810, APC-830 for red laser. These were created by covalently linking a protein donor dye with an organic small molecule acceptor dye. Then, the fluorochromes were conjugated with antibodies using Click chemistry, which is a simple, robust reaction with high-yield and high-reaction sensitivity. Also, we demonstrated long-term stability at −20°C with protein stabilizing cocktails. Most importantly, in order to show the utility of these novel fluorochromes, we created a 34-color flow cytometry panel to measure broad human immune function with high sensitivity on a 3-laser Aurora. The additional UV laser upgrade will increase the instrument capacity to more than 40 color.
This panel will be applied to identify the expression patterns of many different receptors involved in trafficking on multiple different cell types from dissociated tumors and blood samples. In conclusion, this high-content flow cytometry panel will improve analysis and diagnosis in Immuno-oncology and facilitate innovation in biomarker discovery.
Recent advances in flow cytometry instrumentation and fluorochrome chemistries have greatly increased fluorescent conjugated antibody combinations that can be used reliably and easily in routine ...experiments. The Cytek Aurora flow cytometer was first released with three excitation lasers (405, 488, and 640 nm) and incorporated the latest Avalanche Photodiode (APD) technology, demonstrating significant improvement in sensitivity for fluorescent emission signals longer than 800 nm. However, there are limited commercially available fluorochromes capable of excitation with peak emission signals beyond 800 nm. To address this gap, we engineered six new fluorochromes: PE‐750, PE‐800, PE‐830 for the 488 nm laser and APC‐750, APC‐800, APC‐830 for the 640 nm laser. Utilizing the principal of fluorescence resonance energy transfer (FRET), these novel structures were created by covalently linking a protein donor dye with an organic small molecule acceptor dye. Additionally, each of these fluorochrome conjugates were shown to be compatible with fixation/permeabilization buffer reagents, and demonstrated acceptable brightness and stability when conjugated to antigen‐specific monoclonal antibodies. These six novel fluorochrome reagents can increase the numbers of fluorochromes that can be used on a spectral flow cytometer.
Although the T helper 2 (Th2) subset is a critical player in the humoral immune response to extracellular parasites and suppression of Th1-mediated inflammation, Th2 cells have been implicated in ...allergic inflammatory diseases such as asthma, allergic rhinitis, and atopic dermatitis. GATA binding protein 3 (GATA3) is a primary transcription factor that mediates Th2 differentiation and secretion of Th2 cytokines, including IL-4, IL-5, and IL-13. Here, a nucleus-deliverable form of GATA3-transcription modulation domain (TMD) (ndG3-TMD) was generated using Hph-1 human protein transduction domain (PTD) to modulate the transcriptional function of endogenous GATA3 without genetic manipulation. ndG3-TMD was shown to be efficiently delivered into the cell nucleus quickly without affecting cell viability or intracellular signaling events for T cell activation. ndG3-TMD exhibited a specific inhibitory function for the endogenous GATA3-mediated transcription, such as Th2 cell differentiation and Th2-type cytokine production. Intranasal administration of ndG3-TMD significantly alleviated airway hyperresponsiveness, infiltration of immune cells, and serum IgE level in an OVA-induced mouse model of asthma. Also, Th2 cytokine secretion by the splenocytes isolated from the ndG3-TMD-treated mice substantially decreased. Our results suggest that ndG3-TMD can be a new therapeutic reagent to suppress Th2-mediated allergic diseases through intranasal delivery.
•ndG3-TMD is a fusion protein between GATA3-TMD and human-origin Hph-1-PTD.•ndG3-TMD can be transduced into the nucleus of the cells effectively and stably.•ndG3-TMD functions as a competitive inhibitor for GATA3-mediated transcription.•ndG3-TMD inhibits transcription of Th2-type cytokines and Th2 cell differentiation.•Intranasal delivery of ndG3D relieves asthma symptoms in an asthma mouse model.
Abstract
Students entering graduate study in immunology face a sharp transition into the autonomous world of laboratory research and a steep learning curve for the techniques particular to ...immunology. Our goal was to expose new graduate students to essential research skills to facilitate the transition into rotation laboratories and eventually a thesis lab. At Stanford University, over the past three years, we incorporated a weeklong intensive orientation (“Startup”) involving a series of guided, hands-on projects and workshops designed to introduce incoming graduate students to experimental methods in immunology research. Our curriculum posed questions in both mouse and human immunology, answered through pre-validated experiments including flow cytometry, quantitative PCR and fluorescence microscopy. For example, cytokines were measured by qPCR and intracellular staining of T cells. Data visualization and analysis were introduced through exposure to R, Flowjo and Fiji. The students attended lunches with senior graduate students, dinners with faculty, and tours of core facilities. Surveys of participants before and after Startup revealed improvements in experimental/technical confidence. Also, students reported that Startup built class cohesion and facilitated the selection of rotation labs. In summary, we provide a refined recipe for introducing students to immunology research through hands-on experimental modules that mitigate the pedagogical barriers of first-year graduate school.
Antibody secreting cells are generated in regional lymphoid tissues and traffic as plasmablasts (PB) via lymph and blood to target sites for local immunity. The PB, a transiently activated population ...of B cells, are present in low numbers in the peripheral blood of healthy individuals. After acute infection or in setting of active immune responses, however, the frequency of blood PB increases dramatically, peaking 7 to 9 days after immunization or the onset of symptom. PB localized to target tissues undergo further maturation into antibody-secreting plasma cells. For this tissue-specific migration, the homing signature is imprinted on activated lymphocytes during their development by local environmental signals, but the trafficking mechanisms of PB remain poorly characterized. Here, we used multi-parameter flow cytometry to define trafficking programs (TPs, combinations of adhesion molecules and chemoattractant receptors) of PB, and their imprinting in patients in response to localized infection or immune insults. Using single cell-based analysis, we show that TPs enriched after infection or autoimmune inflammation of mucosae correlate with sites of immune response or symptoms, with different TPs imprinted during small intestinal, colon, throat and upper respiratory immune challenge. PB induced after intramuscular or intradermal influenza vaccination, including flu-specific antibody secreting cells, display TPs characterized by the lack of mucosal homing receptors. PB isolated from healthy donors display diverse mucosa-associated TPs, consistent with homeostatic immune activity. Identification of TP signatures of PB may facilitate non-invasive monitoring of organ-specific immune responses.
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