Patent Application
20240402173
IgE-mediated allergic diseases arise when allergen-specific B cells class switch to produce IgE, which then binds to effector mast cells and basophils to promote clinical allergy (doi: 10.1038/nri2273). B cells differentiate into antibody secreting peripheral plasmablasts before becoming plasma cells (doi: 10.3389/fimmu.2012.00078; doi: 10.1016/B978-0-12-397933-9.00014-X). Recent single-cell RNA sequencing suggested that the majority of human peripheral IgE+ B cells may actually be differentiating plasmablasts (doi: 10.1126/science.aau2599).
While well accepted in allergy biology, the study of IgE+ B cells and plasmablasts prove difficult in large part due to the expression of CD23, the low affinity IgE receptor, and binding of IgE by CD23 on the cell surface of most activated B cells. Typically, identification of IgE+ B cells has required either exclusion of all other B cell classes, where IgM−/IgD−/IgG−/IgA− B cells are assumed to be IgE+ (doi: 10.1016/j.jaci.2014.03.036; doi: 10.1111/all.13421; and doi: 10.1016/j.jaci.2019.04.001), or intracellular staining of IgE production (doi: 10.1111/all.12679), which prevents live cell functional analysis. The quantification of IgE-expressing B cells in these studies likely overestimated their frequency (doi: 10.1016/j.jaci.2019.04.001). An anti-human IgE monoclonal antibody that can distinguish between membrane expressed and receptor-bound IgE has also been used to label IgE+ human B cells (doi: 10.3390/cells8090994).
IgE+ B cells measured in these ways are increased in humans with atopic dermatitis and food allergy, compared to healthy controls (doi: 10.1016/j.jaci.2014.03.036; doi: 10.1111/all.13421), and may also increase following allergen exposure (doi: 10.3390/cells8090994). However, due to the difficulty of characterizing these cells for analysis, the relationship between allergen exposure and the presence of IgE+ B cells in peripheral blood is incompletely understood. Likewise, rare peripheral IgE+ plasmablasts and plasma cells have been identified but their relationship to clinical allergy is less defined (doi: 10.1126/science.aau2599; doi: 10.1046/j.1365-2249.2002.02025.x). Most prior studies do not differentiate between IgE+ B cells and IgE+ plasmablasts.
Immunoglobulin E (IgE) is the antibody that causes allergic reactions through binding of effector cells and stimulation by specific allergens (doi: 10.1038/nm.2755). Allergen-specific IgE is often used to diagnose allergy development and severity (doi: 10.1586/1744666X.2014.872032; doi: 10.1007/si1882-018-0816-4). Allergen-specific serum IgE has also been used to guide which allergens should be included in allergen immunotherapy (doi: 10.1016/j.anai.2013.07.005) and to predict treatment effectiveness (doi: 10.1007/s11882-018-0816-4). However, the concentration of allergen-specific IgE in circulation can be low and the identification of causal allergens is difficult due in part to cross reactivity between major and minor allergens (doi: 10.1111/cea.13432). Additionally, total IgE levels may not predict treatment success, for example as was seen in an Omalizumab trial (doi: 10.1016/j.rmed.2007.01.011). Due to this variability, allergic patients can be surprised by unexpected allergic reactions to new allergens or left unsure of the true disease severity.
IgE is produced when allergen-specific B cells are activated by allergen in the presence of the cytokines IL-4 and IL-13 (doi: 10.1084/jem.168.3.853; doi: 10.1073/pnas.90.8.3730), leading to class switch recombination of the constant region to express the IGHE gene (doi: 10.1016/s0165-2427(01)00355-5; doi: 10.1016/j.jaci.2015.07.014). These class-switched B cells, now expressing an IgE B cell receptor, then receive additional survival signals and begin differentiating into memory B cells and plasma cells, the latter secrete high concentrations of IgE into circulation (doi: 10.1016/j.immuni.2006.12.006; doi: 10.7554/eLife.21238; doi: 10.1126/science.aau2599). During the activation process, some IgE+ B cells differentiate into IgE+ plasmablasts. IgE+ plasmablasts secrete antibody and enter peripheral blood, providing a snapshot of the IgE+ B cell to plasma cell differentiation process simultaneously occurring in the lymph node or local tissue. Peripheral IgE+ plasmablasts may continue to the bone marrow where they further differentiate into plasma cells (doi: 10.1038/nri3795).
Different mammalian species can experience naturally occurring IgE-mediated allergic diseases, including humans, horses, dogs, and cats. The horse has been used as a model for human allergy due to the many similarities in the immune mechanism of disease (doi: 10.1016/j.vetimm.2012.02.007; doi: 10.1016/j.molimm.2021.04.013). Horses also allow the ability to control key variables, such as uniform environment and allergen exposure, identical living conditions for allergic and control animals, and identical treatment for allergic animals, if any. Horses express CD23 on peripheral B cells, and previous work has identified that most equine CD23+ B cells are either IgM+ or IgG+ B cells (doi: 10.1016/j.vetimm.2012.02.007).
High CD23 expression on IgE+ plasmablasts has been first identified by studies in horses. Recent single cell sequencing of human B cell surface proteins identified that some human individuals have two distinct B cell populations based on CD23 expression (CD23lo and CD23hi), suggesting that CD23hi IgE+ plasmablasts also exist in humans (doi: 10.1038/s42003-020-1075-1). This also supports the thought that heterogeneity in CD23 function on B cells and plasmablasts is conserved amongst mammals, the only species which produce IgE. However, the cause and clinical relevance of these two populations were not explored in humans. Similarly, another recent study noted that allergic people have CD23hi class switched B cells that are absent in healthy controls (doi: 10.1111/all.14288).
No additional studies identify or measure IgE+ plasmablasts in species prone to allergy. Likewise, there are no biomarkers to determine whether allergen immunotherapy treatment is effectively desensitizing the individual.
The present disclosure is directed to a method of detecting IgE+ plasmablasts in a mammal, a method of predicting clinical allergy in a mammal, a method of determining the severity of clinical allergy in a mammal, a method for monitoring progression of an allergy in a mammal, a method for monitoring the efficacy of an allergy treatment regimen in a mammal, and a kit for the use of detecting IgE+ plasmablasts in a mammal.
In a first aspect, the present disclosure is directed to a method of detecting IgE+ plasmablasts in a mammal, the method comprising: (i) contacting peripheral blood leukocytes (PBL) or peripheral blood mononuclear cells (PBMC) isolated from a blood sample of the mammal, with a wash solution, followed by contacting the washed PBL or PBMC with an acidic wash solution, and thereafter collecting the cells; then (ii) contacting the collected cells from step (i) with a detection agent directed to IgE; and (iii) detecting IgE+ cells as IgE+ plasmablasts. In some embodiments, the PBL or PBMC are first washed with a wash solution at a pH range of 4-6. In some embodiments, steps (ii) and (iii) comprise (ii) contacting the collected cells from step (i) with a detection agent directed to CD23 and a detection agent directed to IgE; and (iii) detecting CD23+ and IgE+ cells as IgE+ plasmablasts. In some embodiments, the acidic wash solution has a pH in the range of 2.5-3.1. In some embodiments, the acidic wash solution has a pH in the range of 2.8-3.0. In some embodiments, the acidic wash solution has a pH of about 3.0. In some embodiments, the cells collected from step (i) are contacted with the acidic wash solution for about 3-10 minutes. In some embodiments, the acidic wash solution is a lactic acid solution. In some embodiments, the pH is neutralized before the cells are collected. In some embodiments, the detection agent directed to CD23 is a monoclonal antibody, and the detection agent directed to IgE is a monoclonal antibody. In some embodiments, the detection is achieved by a flow cytometer. In some embodiments, the PBLs or PBMCs are obtained by removing red blood cells from a blood sample of the mammal. In some embodiments, the mammal is a human, a horse, a dog, or a cat. In some embodiments, the method of detecting IgE+ plasmablasts in a mammal, further includes determining the percentage of IgE+ cells in the total CD23+ cells.
In some embodiments, the disclosure is directed to a method of quantifying IgE, the method comprising:
In some embodiments, the culturing is performed for a period of about 6 hours to about 96 hours. In some embodiments, the measuring is achieved by a laminar flow device, ELISA, bead-based assay, or similar assay platforms. In some embodiments, the mammal is a human, a horse, a dog, or a cat. In some embodiments, the level of secreted IgE reflects the percentage of CD23+ and IgE+ cells in the total CD23+ cells.
Another aspect of the disclosure is directed to a method of predicting clinical allergy in a mammal, the method comprising detecting IgE+ plasmablasts in the mammal, and determining the risk of developing clinical allergy based on the level of IgE+ plasmablasts. In some embodiments, the IgE+ plasmablasts are detected using the methods disclosed herein. In some embodiments, the level of IgE+ plasmablasts is determined based on the percentage of CD23+ and IgE+ cells in the total CD23+ cells. In some embodiments, the mammal will likely develop clinical allergy if the percentage of CD23+ and IgE+ cells in the total CD23+ cells is above a range of 10% to 14.%. In some embodiments, the level of IgE+ plasmablasts is determined based on the percentage of IgE+ cells in the total sample. In some embodiments, the mammal will likely develop clinical allergy if the percentage of IgE+ cells in total cells is above a range of 0.1-0.15%. In some embodiments, the method further comprises treating the mammal with antihistamines, allergen immunotherapy, or biologics (i.e. monoclonal antibodies) aimed to desensitize the individual to allergens when it is determined that the mammal will likely develop clinical allergy. In some embodiments, the method further comprises treating the mammal by dialyzing the IgE+ CD23+ cells isolated from the mammal. In some embodiments, the mammal is a human, a horse, a dog, or a cat.
Another aspect of the disclosure is directed to a method of predicting clinical allergy in a mammal, the method comprising detecting secreted IgE in the mammal, and determining the risk of developing clinical allergy based on the level of secreted IgE. In some embodiments, the secreted IgE is detected using the methods disclosed herein. In some embodiments, the mammal will likely develop clinical allergy if the level of secreted IgE is above a predetermined threshold level. In some embodiments, the method further comprises treating the mammal with antihistamines, allergen immunotherapy, or biologics (i.e. monoclonal antibodies) aimed to desensitize the individual to allergens when it is determined that the mammal will likely develop clinical allergy. In some embodiments, the mammal is a human, a horse, a dog, or a cat.
In another aspect, the disclosure is directed to a method of determining the severity of clinical allergy in a mammal, comprising detecting IgE+ plasmablasts in the mammal, and determining the severity of clinical allergy based on the level of IgE+ plasmablasts. In some embodiments, the IgE+ plasmablasts are detected using the methods disclosed herein. In some embodiments, the level of IgE+ plasmablasts is determined based on the percentage of CD23+ and IgE+ cells in the total CD23+ cells. In some embodiments, the mammal will have more severe allergy based on an increase in the percentage of CD23+ and IgE+ cells in the total CD23+ cells above a range of 10% to 14.%. In some embodiments, the level of IgE+ plasmablasts is determined based on the percentage of IgE+ cells in the total sample. In some embodiments, the mammal will likely develop more severe allergy based on an increase in the percentage of IgE+ cells in total cells above a range of 0.1-0.15%. In some embodiments, the method further comprises treating the mammal with antihistamines, allergen immunotherapy, or biologics aimed to desensitize the individual to allergens when it is determined that the mammal will likely develop clinical allergy. In some embodiments, the method further comprises treating the mammal by dialyzing the IgE+ CD23+ cells isolated from the mammal. In some embodiments, the mammal is a human, a horse, a dog, or a cat.
Another aspect of the disclosure is directed to a method of determining the severity of clinical allergy in a mammal, comprising detecting secreted IgE in the mammal, and determining the severity of clinical allergy based on the level of secreted IgE. In some embodiments, the secreted IgE is detected using the methods disclosed herein. In some embodiments, the mammal will have more severe allergy based on an increase in the level of secreted IgE that surpasses a predetermined threshold. In some embodiments, the method further comprises treating the mammal with antihistamines, allergen immunotherapy, or biologics aimed to desensitize the individual to allergens when it is determined that the mammal will likely develop clinical allergy. In some embodiments, the mammal is a human, a horse, a dog, or a cat.
Another aspect of the disclosure is directed to a method for monitoring progression of an allergy comprising monitoring the level of IgE+ plasmablasts in a mammal, and determining that allergy is progressing based on an increase in the level of IgE+ plasmablasts. In some embodiments, the IgE+ plasmablasts are detected using methods disclosed herein. In some embodiments, the level of IgE+ plasmablasts is determined based on the percentage of CD23+ and IgE+ cells in the total CD23+ cells. In some embodiments, the level of IgE+ plasmablasts is determined based on the percentage of IgE+ cells in the total sample. In some embodiments, the mammal is a human, a horse, a dog, or a cat.
Another aspect of the disclosure is directed to a method for monitoring progression of an allergy comprising monitoring the level of secreted IgE in a mammal, and determining that allergy is progressing based on an increase in the level of secreted IgE. In some embodiments, the secreted IgE is detected using methods disclosed herein. In some embodiments, the mammal is a human, a horse, a dog, or a cat.
One aspect of the disclosure is directed to a method for monitoring the efficacy of a treatment regimen for allergy, comprising monitoring the level of IgE+ plasmablasts in a mammal undergoing the treatment regimen for allergy, and determining the efficacy of the treatment regimen based on the level of IgE+ plasmablasts, wherein a decrease in the level of IgE+ plasmablasts indicates that the treatment regimen is effective. In some embodiments, the IgE+ plasmablasts are detected using methods disclosed herein. In some embodiments, the level of IgE+ plasmablasts is determined based on the percentage of CD23+ and IgE+ cells in the total CD23+ cells. In some embodiments, the level of IgE+ plasmablasts is determined based on the percentage of IgE+ cells in the total sample. In some embodiments, the mammal is a human, a horse, a dog, or a cat.
Another aspect of the disclosure is directed to a method for monitoring the efficacy of a treatment regimen for allergy, comprising monitoring the level of secreted IgE in a mammal undergoing the treatment regimen for allergy, and determining the efficacy of the treatment regimen based on the level of secreted IgE, wherein a decrease in the level of secreted IgE indicates that the treatment regimen is effective. In some embodiments, the secreted IgE is detected using methods disclosed herein. In some embodiments, the mammal is a human, a horse, a dog, or a cat.
Another aspect of the disclosure is directed to a kit for detecting IgE+ plasmablasts, comprising an acidic wash solution, a monoclonal anti-CD23 antibody, and a monoclonal anti-IgE antibody. In some embodiments, the kit further comprises instructions for use. In some embodiments, the anti-CD23 antibody and the anti-IgE antibody are of different species. In some embodiments, the anti-CD23 antibody is against horse CD23 and the anti-IgE antibody is against horse IgE. In some embodiments, the anti-CD23 antibody is against human CD23 and the anti-IgE antibody is against human IgE. In some embodiments, the kit further comprises labeled detection antibodies against the anti-CD23 antibody and the anti-IgE antibody monoclonal. In some embodiments, the monoclonal anti-CD23 antibody and the monoclonal anti-IgE antibody are coupled to different fluorescent dyes.
One aspect of the current disclosure is directed to IgE+ plasmablasts as a diagnostic biomarker for monitoring individuals at risk for developing allergy, such as those with a genetic predisposition. In such an aspect, the biomarker can be measured to determine whether an allergy is developing, or not, in individuals at risk for developing allergy. For example, these could be children with one or both parents allergic. In some embodiments, the biomarker is measured twice, once when there is no allergen exposure to set a normal baseline for the individual, and once during allergen exposure to determine if the biomarker is elevated. For seasonal allergies, the first measurement occurs outside of the allergy season and the second measurement should during the allergy season. For food allergies, the subject fasts from potential allergens before the first measurement and the potential allergens are introduced before the second measurement.
Another aspect of the current disclosure is directed to IgE+ plasmablasts as a diagnostic biomarker as a treatment success indicator for allergen immunotherapy or other biological allergy treatments. Before beginning therapy, the biomarker is measured 1-2 times to create a normal baseline for the individual. If possible, measure the biomarker when the individual is experiencing allergic signs and when the individual is in allergy remission to establish the biomarker range and individual's biomarker profile. For example, the biomarker could then be measured approximately one week following each allergen injection to monitor whether the immunotherapy parameters are working effectively to desensitize the individual or not. Injections (allergen type and dose, injection site, frequency of injections) that decrease the biomarker can be established and continued to develop an optimized treatment regime for each patient. Injections that do not change the biomarker should be modified for the individual.
Another aspect of the current disclosure is directed to IgE+ plasmablasts as a diagnostic biomarker to confirm if clinical signs are caused by allergic reactions or other reasons. IgE+ plasmablasts are used as a diagnostic biomarker to diagnose if new clinical signs in a subject are due to an allergic reaction or not. In this case, the biomarker should be measured as soon as possible during, or immediately (e.g. 1-2 days) following clinical signs. If the biomarker value is above the set threshold for that species, the individual is at risk of being allergic and the clinician can proceed with further tests to diagnose the causal allergen(s). If the biomarker is below the set threshold, a second timepoint can be measured one month later for confirmation and other non-allergy causes should be investigated.
The disclosed addresses three problems that have currently no reliable solution: (1) There are no reliable diagnostic biomarkers to predict the development and progression of allergic diseases. It is not yet possible to determine if an individual is developing allergy before clinical signs become visible. At the time clinical allergy is apparent, the patient's immune system has already dysregulated various immune functions that are not easy to reverse once started. (2) In addition, there is no diagnostic biomarker to monitor the effectiveness of allergen immunotherapy. Allergen immunotherapy is the only current approved allergy treatment that is not just symptomatic but tries to cure and reverse the immune dysregulation leading to allergy. A predictive and diagnostic allergy biomarker can be used to determine if the dosage, timing and site of injections and/or allergen selection is working to desensitize and heal the individual. As a result, allergen immunotherapy is often long-lasting, unsuccessful and expensive. (3) Finally, there are no rapid and repeatable methods to identify and study IgE-secreting cells in the context of allergy, thereby limiting the ability to use these cells as diagnostics. All currently available methods to identify and/or analyze IgE-secreting cells require a complex marker panel which is complicated and lacks repeatability in clinical diagnostic applications.
Currently, no technologies address these three problems. The disclosure herein improves upon several aspects of existing technologies. First, in humans, soluble allergen-specific IgE in serum is used as a diagnostic marker. However, allergen-specific IgE concentrations are low in serum and can vary due to the short half-life of IgE in the circulation. This makes the threshold of detection problematic to define. Both false negative and false positive reactions often occur by using tests that detect allergen-specific IgE in serum. The use of IgE as a biomarker also depends on identification of the specific allergens that an individual is allergic to and is often not consistent in all individuals. In horses and other veterinary species, major allergens are not always defined. Thus, allergen-specific IgE detection is challenging due to the lack of available reagents and/or low concentration in serum. Total serum IgE has sometimes been evaluated instead and is not a consistent biomarker. This invention circumvents these challenges by using the cellular source of IgE (IgE+ plasmablasts) as the biomarker, instead of the product (soluble serum IgE).
Second, IgE+ plasmablasts have been identified in humans, however the experimental approach to identify IgE+ plasmablasts is complex, difficult to analyze, and challenging to repeat. Various research groups have worked on this task and have not been able to clearly identify and distinguish IgE secreting cells from IgG secreting cells. This is due to CD23-bound IgE on the surface of IgE+ plasmablasts. IgE+ plasmablasts, unlike other plasmablasts, uniquely express CD23, previously preventing the distinction from other B cells, which all also express CD23. Therefore, difficult approaches, often based on multiple cell surface markers, had to be designed to address this problem. The disclosed method circumvents this challenge by using a novel acid wash approach to remove CD23-bound IgE and enable simple and rapid quantification of IgE+ plasmablasts in peripheral blood.
The disclosure provides a solution to each of these problems by identifying a novel predictive biomarker that 1) is detectable before clinical allergy, 2) is dynamic and responds quickly to environmental changes, such as allergen exposure and treatment, and 3) can be rapidly and quantitatively measured with the disclosed acid wash method (
In one aspect, the disclosure is directed to a method of detecting IgE+ plasmablasts in a mammal. In some embodiments, the methods comprise a first step (i) of contacting peripheral blood leukocytes (PBL) or peripheral blood mononuclear cells (PBMC) isolated from a blood sample of the mammal, with an acidic wash solution, and thereafter collecting the cells. In some embodiments, the methods comprise a second step (ii) of contacting the collected cells from step (i) with a detection agent directed to IgE. In some embodiments, the methods comprise a third step (iii) of detecting IgE+ cells as IgE+ plasmablasts (
As used herein, “plasmablasts” are plasma cell precursors which undergo cell division, unlike fully differentiated plasma cells. Plasmablasts produce various immunoglobulin isotypes. One such isotype is IgE. Immunoglobulin E (IgE) is the antibody that causes allergic reactions through binding of effector cells and stimulation by specific allergens. IgE is produced when allergen-specific B cells are activated by allergen in the presence of the cytokines IL-4 and IL-13, leading to class switch recombination of the constant region to express the IGHE gene. These class-switched B cells, now expressing an IgE B cell receptor, then receive additional survival signals and begin differentiating into memory B cells and plasma cells, which secrete high concentrations of (soluble) IgE into circulation. During this activation process, some IgE+ B cells differentiate into IgE+ plasmablasts. IgE+ plasmablasts secrete antibody and enter peripheral blood, providing a snapshot of the IgE+ B cell to plasma cell differentiation process simultaneously occurring in the lymph node or local tissue. Peripheral IgE+ plasmablasts may continue to the bone marrow where they further differentiate into plasma cells. Plasmablasts are characterized by both Ig surface expression and secretion. As used herein, “IgE+ plasmablasts” are cells which express IgE on the cell surface and secrete IgE. IgE+ plasmablasts can be found in peripheral blood.
CD23, a C-type lectin expressed on the surface of activated B cells in mammals, is a low affinity IgE receptor. CD23 has different roles in B cell signaling and is most often assumed to decrease IgE production and subsequent allergy. IgE binding to CD23 decreases the availability of IgE for sensitization on mast cells and basophils, and also downregulates IgE production. However, CD23 on B cells is also associated with the facilitation of antigen presentation. During antigen presentation, allergen/IgE complexes are internalized and presented on B cell MHCII molecules, or transferred to dendritic cells, to promote epitope spreading and T cell activation, thereby promoting allergic responses. While CD23 is expressed on most activated peripheral B cells, recent single cell sequencing of B cell surface proteins identified that some human individuals have two distinct CD23+ B cell populations (CD23lo and CD23hi). This suggests heterogeneity in CD23 function on B cells. However, the cause and clinical relevance of these two populations was not explored. Similarly, another recent study noticed that allergic humans have CD23hi class switched B cells that are absent in healthy controls. However, due to CD23-bound IgE, this unique CD23hi B cell population was not further characterized. In addition, increased CD23 surface density on B cells has been positively correlated with allergen-specific IgE levels in allergic individuals, further supporting that variations in B cell CD23 expression may occur in the context of allergy. This suggests that CD23hi cells may have clinical relevance during allergic diseases. As used herein, “CD23+ B cells” refers to B cells expressing CD23 on the cell surface.
As used herein, “peripheral blood leukocytes” or “PBL” are a group of closely related cells, including neutrophils, monocytes, eosinophils, basophils, and lymphocytes. These cells continuously move throughout the body by means of the blood stream, lymphatics, and their ability to migrate through tissues. In some instances, PBL are commonly known as white blood cells. As used herein, “peripheral blood mononuclear cells” or “PBMC” are identified as any blood cell with a round nucleus, i.e. lymphocytes, monocytes, natural killer cells (NK cells) or dendritic cells. PBMC are isolated from peripheral blood. Methods of isolating PBL and PBMC are known in the art. In some embodiments, methods of isolating PBL and PBMC occur through methods explained below. In some embodiments, the PBLs or PBMCs are obtained by removing red blood cells from a blood sample of the mammal. In humans, this can be done by a short hypotonic lysis step or by using an erythrocyte separator tube. In horses, heparinized peripheral blood can be left in collection tubes for approximately 30 minutes at room temperature to allow the erythrocytes to settle and leukocyte-rich plasma (peripheral blood leukocytes, PBL) can be collected. In some embodiments, samples do not need to be processed in a sterile biosafety cabinet.
In some embodiments, the acidic wash solution has a pH of 3.1 or lower. In some embodiments, the acidic wash solution has a pH range of pH 2.5-3.1. In some embodiments, the acidic wash solution has a pH in the range of pH 2.8-3.0. In some embodiments, the acidic wash solution has pH of about pH 3.0 or less. In some embodiments, the acidic wash solution is any acidic solution having a pH of about 3.1 or less. In some embodiments, the acidic wash solution has pH of about pH 2.9 or less. In some embodiments, the acidic wash solution is any acidic solution having a pH of about 2.8 or less. In some embodiments, the acidic wash solution has pH of about pH 2.7 or less. In some embodiments, the acidic wash solution is any acidic solution having a pH of about 2.6 or less. In some embodiments, the acidic wash solution has pH of about pH 2.5 or less. In some embodiments, the acidic wash solution is a lactic acid. In some embodiments, the acidic wash solution is a citric acid. In some embodiments, the acidic wash solution is a hydrochloric acid. In some embodiments, the acidic wash solution may be a combination of acids.
In some embodiments, the isolated PBL or PBMC are contacted with the acidic wash solution for about 3-10 minutes. In some embodiments, the isolated PBL or PBMC are contacted with the acidic wash solution for about 10 minutes. In some embodiments, the isolated PBL or PBMC are contacted with the acidic wash solution for about 9 minutes. In some embodiments, the isolated PBL or PBMC are contacted with the acidic wash solution for about 8 minutes. In some embodiments, the isolated PBL or PBMC are contacted with the acidic wash solution for about 7 minutes. In some embodiments, the isolated PBL or PBMC are contacted with the acidic wash solution for about 6 minutes. In some embodiments, the isolated PBL or PBMC are contacted with the acidic wash solution for about 5 minutes. In some embodiments, the isolated PBL or PBMC are contacted with the acidic wash solution for about 4 minutes. In some embodiments, the isolated PBL or PBMC are contacted with the acidic wash solution for about 3 minutes.
In some embodiments, the isolated PBL or PBMC are washed with a wash solution prior to the step of contacting them with an acidic wash solution. In some embodiments, the wash solution is not a buffered solution. In some embodiments, the wash solution is at a pH below 7.0. In some embodiments, the wash solution is pH 6.8 or lower. In some embodiments, the wash solution is pH 6.6 or lower. In some embodiments, the wash solution is pH 6.4 or lower. In some embodiments, the wash solution is pH 6.2 or lower. In some embodiments, the wash solution is pH 6.1 or lower. In some embodiments, the wash solution is pH 6.0 or lower. In some embodiments, the wash solution is pH 5.9 or lower. In some embodiments, the wash solution is pH 5.8 or lower. In some embodiments, the wash solution is pH 5.7 or lower. In some embodiments, the wash solution is pH 5.6 or lower. In some embodiments, the wash solution is pH 5.5 or lower. In some embodiments, the wash solution is pH 5.4 or lower. In some embodiments, the wash solution is pH 5.2 or lower. In some embodiments, the wash solution is pH 5.0 or lower.
In some embodiments, steps (ii) and (iii) comprise (ii) contacting the collected cells from step (i) with a detection agent directed to IgE; and (iii) detecting IgE+ cells as IgE+ plasmablasts. In some embodiments, the detection agent directed to IgE is a monoclonal antibody. In some embodiments, the detection of IgE+ cells is achieved through flow cytometry.
In some embodiments, steps (ii) and (iii) comprise (ii) contacting the collected cells from step (i) with a detection agent directed to CD23 and a detection agent directed to IgE; and (iii) detecting CD23+ and IgE+ cells as IgE+ plasmablasts. In some embodiments, the detection agent directed to CD23 is a monoclonal antibody. In some embodiments, the detection agent directed to IgE is a monoclonal antibody. In some embodiments, the detection of CD23+ and IgE+ cells is achieved through flow cytometry.
In some embodiments, different fluorochromes are conjugated to the two monoclonal antibodies to fit the available flow cytometer. Fluorochromes that are excited by two different lasers, if possible, can be used to eliminate the need for compensation. Alexa fluorochromes were used herein due to the simplicity of the manufacturer's protocol for antibody-fluorochrome conjugation, and the slow bleach rate of the dye. However, other fluorochromes can be used.
In some embodiments, the PBL or PMBC are then incubated for flow cytometry in PBS-BSA with two monoclonal antibodies (anti-IgE, e.g., conjugated to Alexa fluorochrome 647, and anti-CD23, e.g., conjugated to Alexa fluorochrome 488). Cells are incubated for 15 minutes at room temperature in the dark and then washed once with 1 ml PBS-BSA. Samples are recorded on a flow cytometer. To analyze, flow cytometry data image is gated first to exclude doublets (FSC-A x FSC-H plot, gate selects cells along the diagonal) and second to gate on CD23+ cells (gate selects cells in the right half of a plot showing CD23 on the x axis and SSC-A on the y axis). A third gate is applied to cells in the upper right quadrant of a CD23 (x axis) x IgE (y axis) plot (
In some embodiments, cells are incubated with only an anti-IgE monoclonal antibody. In some embodiments, the monoclonal antibody is fluorescently conjugated. In some embodiments, analysis first excludes doublets (FSC-A x FSC-H plot, gate on cells along the diagonal) and second gate on IgE+ cells (gate selects cells in the right half of a plot showing IgE on the x axis and SSC-A on the y axis). The percentage of IgE+ out of total doublet-excluded events would be the diagnostic readout (
In some embodiments, the pH is neutralized after the PBL or PBMC are contacted with the acidic wash solution but before collecting the cells. In some embodiments, the pH is neutralized after the acidic wash solution by using Tris-HCl buffer solution at pH7-8. In some embodiments, the pH is neutralized with PBS. In some embodiments, the pH is neutralized with another similar buffered solution with a neutral pH.
As used herein, “mammal” is any mammal. In some embodiments, the mammal is a human, horse, dog, or cat. As used herein, “subject” is any mammal from which the blood sample has been obtained.
In some embodiments, the percentage of IgE+ cells in the total CD23+ cells is determined.
Some embodiments of the disclosure are directed to methods of quantifying IgE+ plasmablasts, the methods comprising culturing peripheral blood leukocytes (PBL), or peripheral blood mononuclear cells (PBMC) isolated from a blood sample of a mammal, measuring IgE secreted from the cultured cells in the supernatant; and correlating the level of secreted IgE with the level of IgE+ plasmablasts. In some embodiments, the secreted IgE is measured by a laminar flow device, ELISA, bead-based assays, or similar assay platforms known in the art. In some embodiments, the level of IgE+ plasmablasts reflects the percentage of IgE+ cells in the total CD23+ cells.
In some embodiments, the PBL or PBMC are cultured for a period of about 6 hours to about 96 hours. In some embodiments, the PBL or PBMC are cultured for a period of about 12 hours to about 90 hours. In some embodiments, the PBL or PBMC are cultured for a period of about 18 hours to about 84 hours. In some embodiments, the PBL or PBMC are cultured for a period of about 24 hours to about 72 hours.
In some embodiments, secreted IgE from IgE+ plasmablasts is measured as a quantitative readout. In some embodiments, the disclosure is directed to methods of quantifying secreted IgE comprising culturing PBL or PBMC isolated from a blood sample of a mammal, measuring IgE secreted from the cultured cells in the supernatant, and quantifying the level of secreted IgE. In some embodiments, secreted IgE directly correlates to the frequency of IgE+ plasmablasts in peripheral blood (see
In some embodiments, an ELISpot assay is used to measure the frequency of IgE-secreting plasmablasts in a subject as a quantitative readout. In some embodiments, plates are coated with allergen or anti-IgE antibodies, then PBL or whole blood samples added to the plate and incubated. This allows all IgE+ plasmablasts to secrete IgE, which binds to the anti-IgE antibody (or allergen). Secreted IgE could then be quantified in spots to determine the frequency of IgE+ plasmablasts.
In some embodiments, the disclosed acid wash approach is rapid, providing a turn-around time of 2 hours from retrieval of the sample to quantitative result. In some embodiments, the methods can be performed in a non-sterile environment. In some embodiments, the methods require minimal flow cytometer capacity or user technical background.
A further aspect of the disclosure is directed to a method of predicting clinical allergy in a mammal, the method comprising detecting IgE+ plasmablasts in the mammal, and determining the risk of developing clinical allergy based on the level of IgE+ plasmablasts. In some embodiments, the IgE+ plasmablasts are detected using the methods disclosed herein. In some embodiments, the level of IgE+ plasmablasts is determined based on the percentage of CD23+ and IgE+ cells in the total CD23+ cells. In some embodiments, the level of IgE+ plasmablasts is determined based on the percentage of IgE+ cells in the total sample. In some embodiments, the disclosure is directed to a method of determining the severity of clinical allergy in a mammal, comprising detecting IgE+ plasmablasts in the mammal, and determining the severity of clinical allergy based on the level of IgE+ plasmablasts.
In some embodiments, the mammal will likely develop clinical allergy if the percentage of IgE+ cells in the total CD23+ cells is above 10%. In some embodiments, the mammal will likely develop clinical allergy if the percentage of IgE+ cells in the total CD23+ cells is above 11%. In some embodiments, the mammal will likely develop clinical allergy if the percentage of IgE+ cells in the total CD23+ cells is above 12%. In some embodiments, the mammal will likely develop clinical allergy if the percentage of IgE+ cells in the total CD23+ cells is above 13%. In some embodiments, the mammal will likely develop clinical allergy if the percentage of IgE+ cells in the total CD23+ cells is above 14.%.
In some embodiments, the mammal will likely develop clinical allergy if the percentage of IgE+ cells in the total cells is above 0.1%. In some embodiments, the mammal will likely develop clinical allergy if the percentage of IgE+ cells in the total cells is above 0.11%. In some embodiments, the mammal will likely develop clinical allergy if the percentage of IgE+ cells in the total cells is above 0.12%. In some embodiments, the mammal will likely develop clinical allergy if the percentage of IgE+ cells in the total cells is above 0.13%. In some embodiments, the mammal will likely develop clinical allergy if the percentage of IgE+ cells in the total cells is above 0.14%. In some embodiments, the mammal will likely develop clinical allergy if the percentage of IgE+ cells in the total cells is above 0.15%.
In some embodiments, the method further comprises treating the mammal with antihistamines, allergen immunotherapy, or biologics such as monoclonal antibodies or cell-based therapies aimed to desensitize the individual to allergens when it is determined that the mammal will likely develop clinical allergy.
In some embodiments, the method further comprises treating the mammal by dialyzing the IgE+ CD23+ cells isolated from the mammal. In some embodiments, the mammal is a human, a horse, a dog, or a cat. In some embodiments, the dialysis runs through a low pH chamber to remove surface-bound IgE, followed by a chamber with an anti-IgE monoclonal antibody. A threshold to remove cells fluoresce due to that antibody would be set to selectively remove IgE+ cells. The acid wash chamber would prevent accidental removal of normal CD23+ B cells. In some embodiments, subjects with autoimmune disease undergo dialysis to remove IgE+ cells.
Another aspect of the disclosure is directed to a method of predicting clinical allergy in a mammal, the method comprising detecting secreted IgE in the mammal, and determining the risk of developing clinical allergy based on the level of secreted IgE. In some embodiments, the secreted IgE is detected using the methods disclosed herein. In some embodiments, the disclosure is directed to a method of determining the severity of clinical allergy in a mammal, comprising detecting secreted IgE in the mammal, and determining the severity of clinical allergy based on the level of secreted IgE.
In some embodiments, the mammal will likely develop clinical allergy if the level of secreted IgE passes a predetermined threshold. In some embodiments, the predetermined threshold is a detected level of secreted IgE above 200 ng/mL. In some embodiments, the predetermined threshold is a detected level of secreted IgE above 300 ng/mL. In some embodiments, the predetermined threshold is a detected level of secreted IgE above 400 ng/mL. In some embodiments, the predetermined threshold is a detected level of secreted IgE above 500 ng/mL. In some embodiments, the predetermined threshold is a detected level of secreted IgE above 600 ng/mL. In some embodiments, the predetermined threshold is a detected level of secreted IgE above 700 ng/mL. In some embodiments, the predetermined threshold is a detected level of secreted IgE above 800 ng/mL. In some embodiments, the predetermined threshold is a detected level of secreted IgE above 900 ng/mL. In some embodiments, the predetermined threshold is a detected level of secreted IgE above 1,000 ng/mL. In some embodiments, the predetermined threshold is a detected level of secreted IgE above 1,500 ng/mL.
In some embodiments, the method further comprises treating the mammal with antihistamines, allergen immunotherapy, or biologics such as monoclonal antibodies or cell-based therapies aimed to desensitize the individual to allergens when it is determined that the mammal will likely develop clinical allergy.
Another aspect of the disclosure is directed to a method for monitoring progression of an allergy comprising monitoring the level of IgE+ plasmablasts in a mammal, and determining that allergy is progressing based on an increase in the level of IgE+ plasmablasts. In some embodiments, the IgE+ plasmablasts are detected using methods disclosed herein. In some embodiments, the level of IgE+ plasmablasts is determined based on the percentage IgE+ cells in the total CD23+ cells. In some embodiments, the level of IgE+ plasmablasts is determined based on the percentage of IgE+ cells in the total sample. In some embodiments, the mammal is a human, a horse, a dog, or a cat.
In some embodiments, the progression of an allergy is based on monitoring the efficacy of a treatment regimen for the allergy, comprising monitoring the level of IgE+ plasmablasts in a mammal undergoing the treatment regimen for allergy, and determining the efficacy of the treatment regimen based on the level of IgE+ plasmablasts. In some embodiments, a decrease in the level of IgE+ plasmablasts indicates that the treatment regimen is effective. In some embodiments, the IgE+ plasmablasts are detected using methods disclosed herein. In some embodiments, the level of IgE+ plasmablasts is determined based on the percentage of CD23+ and IgE+ cells in the total CD23+ cells. In some embodiments, the level of IgE+ plasmablasts is determined based on the percentage of IgE+ cells in the total sample. In some embodiments, the mammal is a human, a horse, a dog, or a cat.
Another aspect of the disclosure is directed to a method for monitoring progression of an allergy comprising monitoring the level of secreted IgE in a mammal, and determining that allergy is progressing based on an increase in the level of secreted IgE. In some embodiments, the secreted IgE is detected using methods disclosed herein. In some embodiments, the mammal is a human, a horse, a dog, or a cat.
In some embodiments, the progression of an allergy is based on monitoring the efficacy of a treatment regimen for the allergy, comprising monitoring the level of secreted IgE in a mammal undergoing the treatment regimen for allergy, and determining the efficacy of the treatment regimen based on the level of secreted IgE. In some embodiments, a decrease in the level of secreted IgE indicates that the treatment regimen is effective. In some embodiments, the secreted IgE is detected using methods disclosed herein. In some embodiments, the mammal is a human, a horse, a dog, or a cat.
Another aspect of the disclosure is directed to a kit for detecting IgE+ plasmablasts, comprising an acidic wash solution and a monoclonal anti-IgE antibody. In some embodiments, the kit further comprises instructions for use. In some embodiments, the kit further comprises a monoclonal anti-CD23 antibody. In some embodiments, the anti-CD23 antibody and the anti-IgE antibody are of different species. In some embodiments, the anti-CD23 antibody is against horse CD23 and the anti-IgE antibody is against horse IgE. In some embodiments, the anti-CD23 antibody is against human CD23 and the anti-IgE antibody is against human IgE. In some embodiments, the kit further comprises labeled detection antibodies against the anti-CD23 antibody and the anti-IgE antibody monoclonal. In some embodiments, the monoclonal anti-CD23 antibody and the monoclonal anti-IgE antibody are coupled to different color fluorescent beads.
In some embodiments, the kit is for clinical use. In some embodiments, the kit is for research purposes.
Ultimately, the methods described herein provide clear quantitative results that allow for objective interpretation.
Some embodiments of the disclosure are directed to using smaller volumes of leukocyte-rich plasma and still ensuring accurate quantification of IgE+ plasmablasts. In some embodiments, at least 500 μl of leukocyte-rich plasma is used. In some embodiments, at least 400 μl of leukocyte-rich plasma is used. In some embodiments, at least 300 μl of leukocyte-rich plasma is used.
Some embodiments of the disclosure are directed to using smaller volumes of whole blood and still ensuring accurate quantification of IgE+ plasmablasts. In some embodiments, at least 500 μl of whole blood is used. In some embodiments, at least 400 μl of whole blood is used. In some embodiments, at least 300 μl of whole blood is used.
In some embodiments of the disclosure, heparinized blood samples are stored at 4° C. for 1-4 days, or longer, before processing. In some embodiments, when the heparinized blood samples are ready to be processed, blood tubes are gently inverted multiple times before processing the sample. In some embodiments, 1-2 ml of leukocyte-rich plasma is collected and used with the acid wash of the disclosure. In some embodiments, 1-2 ml of whole blood is collected and used with the acid wash protocol of the disclosure.
In some embodiments, erythrocytes are removed from the sample. In humans, this can be done by a short hypotonic lysis step or by using an erythrocyte separator tube. In horses, heparinized peripheral blood can be left in collection tubes for approximately 30 minutes at room temperature to allow the erythrocytes to settle and leukocyte-rich plasma (peripheral blood leukocytes, PBL) can be collected. In some embodiments, samples do not need to be processed in a sterile biosafety cabinet. 1 ml PBL is collected and pelleted at 500×g 10 min at 4° C. All subsequent wash steps are performed at 100 g for 5 min at 4° C. Cells are washed twice with 1 ml ice-cold wash solution (130 mM NaCl, 5 mM KCl; pH 6). Then, cells are resuspended in ice-cold acid wash solution (10 mM lactic acid (Alfa Aesar, Thermo Fisher Scientific, Lancashire, UK), 130 mM NaCl, 5 mM KCl; pH 2.8-3) and incubated for 5 min on ice. After incubation, 0.2 ml of 1 M Tris HCl, pH 8 (Thermo Fisher Scientific, Waltham, MA, USA) is added to neutralize the acid before pelleting the cells by centrifugation. Cells are washed once with 1 ml PBS and fixed in 2% (v/v) paraformaldehyde solution (PFA, Sigma-Aldrich, St. Louis, MO, USA) for 20 minutes at room temperature, then washed twice in 1 ml PBS and once in 1 ml PBS-BSA (0.5% (w/v) BSA, 0.02% (w/v) NaN3, all from Sigma-Aldrich, St. Louis, MO, USA). Wash steps after formalin fixation are performed at room temperature.
In some embodiments, the formalin-fixation step is skipped, and live cells are incubated with antibodies and analyzed. In some such embodiments, steps of the disclosed acid wash methods are performed on ice. In some embodiments, steps of the disclosed acid wash methods are performed on the same day as sample collection. In some embodiments, steps of the disclosed acid wash methods are performed on ice and on the same day as sample collection.
Some embodiments of the disclosure are directed to an allergy treatment. In some embodiments, allergic individuals following allergen exposure undergo a dialysis-like treatment where their peripheral blood B-cells are acid washed and labeled for IgE+ cells. The IgE+ plasmablasts are removed from circulation and the rest of the blood cells are immediately buffered to become isotonic, and replaced into the subject. Such embodiments could decrease the progression of the allergic response through the removal of the source of IgE, interrupting the vicious cycle of IgE production during allergic diseases.
Some embodiments of the disclosure are directed to diagnostic tests measuring secreted IgE. In some embodiments, a rapid secreted IgE assay kit is used for allergy doctors to measure as a correlate for IgE+ plasmablasts. In some embodiments, this occurs in the doctor's office. In some embodiments, a peripheral blood sample is collected and added to an incubation well for an incubation at 37° C. Following incubation, the culture supernatant is harvested and added to a rapid lateral flow device to measure secreted IgE. Secreted IgE correlates directly with the frequency of the IgE+ plasmablast biomarker providing a simple, quick, and reliable quantification method. In some embodiments, the measuring occurs in an outsourced laboratory. In some embodiments, the measuring occurs in a local laboratory. In some embodiments, the measuring occurs in the lab of a doctor's office. In some embodiments, the measuring occurs in the doctor's office.
Some embodiments of the disclosure are directed to research test kits. In some embodiments, the acid wash approach to clearly identify and isolate IgE+ plasmablasts will allow scientific research advancement to study their roles during the pathomechanisms of allergy. In some embodiments, the kit comprises pre-made solutions. In some embodiments, live cells can be stained with the described antibodies and sorted by flow cytometry for functional and/or transcriptomic analysis. In some embodiments, sorted IgE+ plasmablasts are immortalized to generate purified allergen-specific monoclonal IgE. In some embodiments, the immortalized IgE+ plasmablasts are cloned. In some embodiments, the immortalized IgE+ plasmablasts are used to study the mechanisms of IgE+ plasmablasts (i.e. allergen binding and internalization, downstream signaling pathways, etc.).
Some embodiments of the disclosure are directed to allergen-specific diagnostics and allergen discovery. In some embodiments, IgE+ plasmablasts from a subject are purified and used to determine the specific allergen panel the subject is sensitized to. In such embodiments, the allergen-specificity of IgE secreted by IgE+ plasmablasts is measured. Such embodiments may have higher sensitivity than serum IgE measurement.
CD23-bound IgE on B cells and plasmablasts exist as a heterotrimer in complex with allergen. Therefore, some embodiments comprise using the disclosed acid wash for the removal of isolation of IgE/allergen complexes. In some embodiments, purified allergen from the isolated IgE/allergen complexes are characterized by mass spectroscopy to identify candidate allergens.
In some embodiments, the disclosure is directed to autologous IgE+ plasmablast therapy. In some embodiments, isolated IgE+ plasmablasts are used to model an allergic immune response to optimize and guide allergen immunotherapy in a research setting. In some embodiments, autologous IgE+ plasmablasts are harvested from a subject and frozen during clinical allergy. Then the autologous IgE+ plasmablasts are injected to the subject during remission, in the absence of naturally occurring allergens. The immune response following allergen injections during allergy remission is studied. In some embodiments, the injection of autologous IgE+ plasmablasts in the absence of allergen induces a regulatory and desensitization pathway, contributing to a form of immunotherapy.
The following examples are presented to illustrate the present disclosure. The examples are not intended to be limiting in any manner.
Heparinized blood samples settled at room temperature for at least 30 minutes to allow erythrocytes to separate from the cell-rich plasma. Cell rich plasma (1 ml), containing peripheral blood leukocytes (PBL), was collected and centrifuged at 500×g for 10 minutes at 4° C. PBL were then treated with an acid wash. A total of 3×106 PBL were washed in 1 ml ice-cold wash solution (130 mM NaCl, 5 mM KCl; pH 6). Cells were then resuspended in ice-cold acid wash solution (10 mM lactic acid (Alfa Aesar, Thermo Fisher Scientific, Lancashire, UK), 130 mM NaCl, 5 mM KCl; pH 2.8-3) and incubated for 5 min on ice. After incubation, 0.2 ml of 1 M Tris HCl, pH 8 (Thermo Fisher Scientific, Waltham, MA, USA) was added to neutralize the acid before pelleting the cells by centrifugation. All wash steps were performed at 150 g for 5 min at 4° C. Cells were washed once with 1 ml PBS and either used immediately for live cell sorting or fixed in 2% (v/v) paraformaldehyde solution (PFA, Sigma-Aldrich, St. Louis, MO, USA) for 20 minutes at room temperature. An aliquot of PBL without acid washing was fixed in 2% PFA as a control.
All centrifugations were performed at 100×g for 5 minutes at 4° C. Briefly, PBL were washed twice in ice-cold wash solution (130 mM NaCl, 5 mM KCl; pH 6). Cells were then resuspended in ice-cold acid wash solution (10 mM lactic acid (Alfa Aesar, Thermo Fisher Scientific, Lancashire, UK), 130 mM NaCl, 5 mM KCl; pH 2.8-3) and incubated for 5 min on ice. After incubation, 0.2 ml of 1 M Tris HCl, pH 8 (Thermo Fisher Scientific, Waltham, MA, USA) was added to neutralize the acid before pelleting the cells by centrifugation. Cells were washed once in phosphate buffered saline (PBS, Fisher Scientific, Waltham, MA, USA) and then fixed in 2% (v/v) paraformaldehyde solution (PFA, Sigma-Aldrich, St. Louis, MO, USA) for 20 minutes at room temperature.
In addition, peripheral blood mononuclear cells (PBMC) were isolated by layering of cell-rich plasma over Ficoll (Ficoll Plaque Plus, GE Healthcare, Chicago, IL, USA) at a 2:1 (v:v) ratio. Density gradient centrifugation and PBMC isolation was performed. Heparinized peripheral blood was left in collection tubes for approximately 1 hour at room temperature to allow the erythrocytes to settle. The leukocyte-rich plasma was layered on Ficoll (Ficoll Plaque Plus, GE Healthcare, Chicago, IL, USA) at a 2:1 (v:v) ratio, followed by centrifugation at 2000 g for 20 min at 10° C. without brakes. The interphase containing PBMC was collected into a 50 ml conical tube, prefilled with phosphate buffered saline (PBS, Fisher Scientific, Waltham, MA, USA). The pellet was recovered by centrifugation at 500 g for 15 min at room temperature and suspended in PBS. Cells were washed once more in PBS to remove platelets and recovered by centrifugation at 150 g for 5 min at room temperature. All subsequent centrifugations were carried out at 150 g for 5 min at 4° C. or room temperature.
A total of 3×106 PBMC were washed in 1 ml ice-cold wash solution (130 mM NaCl, 5 mM KCl; pH 6). Cells were then resuspended in ice-cold acid wash solution (10 mM lactic acid (Alfa Aesar, Thermo Fisher Scientific, Lancashire, UK), 130 mM NaCl, 5 mM KCl; pH 2.8-3) and incubated for 5 min on ice. After incubation, 0.2 ml of 1 M Tris HCl, pH 8 (Thermo Fisher Scientific, Waltham, MA, USA) was added to neutralize the acid before pelleting the cells by centrifugation. All wash steps were performed at 150 g for 5 min at 4° C. Cells were washed once with 1 ml PBS and either used immediately for live cell sorting or fixed in 2% (v/v) paraformaldehyde solution (PFA, Sigma-Aldrich, St. Louis, MO, USA) for 20 minutes at room temperature. An aliquot of PBMC without acid washing was fixed in 2% PFA as a control.
Following the same PBL isolation as described above, 1 ml cell-rich plasma was collected and centrifuged at 500×g for 10 minutes at 4° C. The cell-depleted plasma was collected and stored at −20° C. until analyzed. PBL were resuspended in cell culture medium (DMEM supplemented with 1% (v/v) non-essential amino acids, 2 mM L-glutamine, 50 μM 2-mercaptoethanol, 50 μg/ml gentamicin, 100 U/ml penicillin, 100 μg/ml streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) and 10% FCS (Atlanta biological, Flowery Branch, GA, USA)), and then centrifuged at 100×g for 5 minutes at room temperature. PBL were resuspended to 1 ml in cell culture medium, and 100 μl of PBL from each individual was mixed with cell culture medium to a final volume of 200 μl and incubated in 96-well flat-bottomed plates (Corning Incorporated, Corning, NY, USA) at 37° C., 5% CO2. After 72-hours, cell-free supernatants were collected and stored at 4° C. until IgE secretion analysis. All steps were performed in a sterile biosafety cabinet.
PBMC were also isolated as described and 5×105 PBMC were plated per well in 200 μl cell culture medium. Cells were incubated as described above for PBL and supernatants were collected for IgE analysis. In some instances, isolated PBMC (6×105 cells/well, containing an average calculated 5.2×102-3.5×103 IgE+ plasmablasts), sorted CD23hi/IgE+ (IgE+ plasmablasts) and CD23lo/IgE− (IgE− B cells) cells (3×103 sorted cells/well) were incubated without stimulation in cell culture medium. Cells were incubated in 96-well flat-bottom plates (Corning Incorporated, Corning, NY, USA) at 37° C., 5% CO2 for 72-hours before cell-free supernatants were collected and stored at 4° C. until analysis.
Secreted equine IgE was measured in undiluted supernatants using a fluorescent bead-based Luminex assay. The assay was set up as previously described with a standard curve of six five-fold dilutions (3.2 ng/ml-10 μg/ml IgE) (doi: published 26). Twelve 1:2 serial dilutions of purified IgE were used as a standard with concentrations ranging from 4.9 ng/ml-10 μg/ml total IgE. Total IgE was also measured in the cell-depleted plasma using eight 1:2 serial dilutions of purified IgE with concentrations ranging from 78.1 ng/ml-10 μg/ml total IgE, allowing measurement of the higher concentration of IgE in serum.
PBMC were stained with different mAbs and analyzed by flow cytometry to further characterize the CD23+ cell population in peripheral blood. CD23+ cells lacked T cell (CD4 and CD8) and monocyte (CD14) surface proteins but expressed high levels of MHCII (
It was assumed that many of the CD23+ cells had bound IgE from circulation to their cell surface via the low affinity IgE-receptor CD23. Most of the IgE could be removed from CD23+ cells by a short lactic acid wash leaving two populations, CD23lo/IgE− and CD23hi/IgE+ (
IgE+ plasmablasts were quantified in peripheral blood by a short lactic acid wash (pH 3), followed by incubation with fluorescent antibodies against IgE and CD23, and quantification by flow cytometry as shown in Example 2. To quantify the IgE+ plasmablast frequency more rapidly in many individuals, this approach was adapted from isolated PBMC (
To further confirm if CD23hi/IgE+ cells produce IgE, CD23-bound IgE was again removed from PBMC by acid wash (
The surface labeling of IgE saturated all surface IgE mAb binding sites and therefore intracellular staining revealed only intracellular IgE. This was confirmed by two sequential extracellular IgE staining steps. The majority of the CD23hi/IgE+ cells were only positive for the first IgE label (
Before becoming plasma cells, B cells differentiate into plasmablasts which secrete antibody and travel from the lymph node through peripheral blood (doi: 10.3389/fimmu.2012.00078; doi: 10.1016/B978-0-12-397933-9.00014-X). To find additional evidence for the differentiation stage of peripheral CD23hi/IgE+ cells as antibody secreting cells, we used the RNA extracted from FACS sorted CD23hi/IgE+ cells and RNA extracted from IgE− B cells sorted simultaneously as controls (
Plasmablasts and plasma cells downregulate transcription factor paired box protein 5 (PAX5) and B cell lymphoma 6 (BCL6) and upregulate B lymphocyte-induced maturation protein 1 (BLIMP-1), interferon regulating factor 4 (IRF4) and x-box binding protein 1 (XBP1) (Table 1). An expression pattern was detected similar to this while comparing the mean difference of expression (ddCt) in CD23hi/IgE+ cells and IgE− B cells. Positive ddCt values represent increased expression in CD23hi/IgE+ cells and vis versa. CD23hi/IgE+ cells had decreased expression of PAX5 (ddCt=−3.67, p=0.144) and BCL6 (ddCt=−4.63, p=0.118) compared to IgE− B cells (
To verify that CD23hi/IgE+ cells had undergone class switching and were truly expressing IgE, expression of the IgE heavy chain constant region (IGHE) RNA transcript was measured by PCR using IGHE-specific primers (Table 1). The PCR product from CD23hi/IgE+ cell cDNA was 1272 base pairs (
Additionally, morphology of CD23hi/IgE+ cells and IgE− B cells were explored by differential staining of sorted cell populations. Consistent with B cell morphology, IgE− B cells had characteristic round, central nuclei with coarsely clumped chromatin, a narrow cytoplasmic area and small cell size (<10 μm diameter;
To determine whether IgE+ plasmablasts were elevated during clinical allergy, percentages were compared of IgE+ plasmablasts in the peripheral blood of healthy horses (n=6) with those of allergic horses with Culicoides (Cul) hypersensitivity (n=7). Cul hypersensitivity is a seasonal allergic disease and therefore allergic horses only develop clinical signs in the summer during Cul allergen exposure (Table 2,
May
onset
c
June
constant
d
5.5
(2.5-6.5)
0.75
(0-1)
July
6
(4-9)
0.5
(0-2)
August
5
(4.5-6)
1
(0.5-2.5)
September
3
(3-4)
0.5
(0-1)
October
decline
e
3
(2-3)
1
(0-1)
However, acid wash treatment of PBMC to remove CD23-bound IgE allows more accurate gating and identification of IgE+ plasmablasts. IgE+ plasmablasts were compared in acid washed PBMC. Allergic horses had significantly increased frequencies of IgE+ plasmablasts compared to healthy horses in July, but not in December when all horses appeared clinically healthy again (
To determine if IgE+ plasmablasts are secreting IgE, isolated PBMC from allergic (n=7) and non-allergic horses (n=7) were cultured for 3 days in cell culture medium without additional stimuli. Afterwards, secreted IgE antibodies were measured in the supernatants. While PBMC from all horses secreted IgE, PBMC from allergic horses secreted significantly more IgE (median 486.2 ng/ml, range 270.8-822.0 ng/ml) than healthy horses (median 149.8 ng/ml, range 94.2-558.5 ng/ml) (
An aliquot of ex vivo PBMC from each horse was treated with the acid wash solution and analyzed by flow cytometry to compare the percentage of IgE+ plasmablasts between groups (
Secreted IgE by IgE+ plasmablasts likely first binds to available IgE receptors, and then contributes to soluble IgE in the cell culture supernatant. To test this, PBMC were acid washed (
IgE+ plasmablasts spontaneously secrete IgE as shown herein. IgE secretion by these cells was measured after 72 hours of in vitro culture of PBL. The concentration of secreted IgE from allergic and healthy horses followed a similar trend as the frequency of IgE+ plasmablasts (
In comparison to the spontaneous IgE secretion from PBL, IgE concentrations in plasma from the same blood samples were measured in all horses. Total IgE in plasma was only elevated in some allergic individuals later in the summer, at the end of the “Chronic Phase” and beginning of the “Resolving Phase”. On July 17, 4 out of 7 allergic horses had plasma IgE concentrations above the plasma IgE threshold of 100 μg/ml. Plasma IgE in 2 of the allergic horses did not cross the threshold until the “Resolving Phase” in October, 5 months after clinical allergy onset. One horse never reached the plasma IgE threshold (
Heparinized blood samples were obtained from the V. jugularis using the BD Vacutainer system (Becton Dickinson, Franklin Lakes, NJ, USA). All animals studied were Icelandic horses living together in the same environment with natural exposure to Cul from mid-May to mid-October. Cul were absent from the environment of the horses for the remainder of the year. Vaccination and deworming were synchronized. All horses were annually vaccinated against rabies, tetanus, West Nile virus and Eastern and Western Encephalitis virus, as well as dewormed with moxidectin and praziquantel (Zoetis, Parsipanny, NJ, USA) once a year in December. All horses were on the same diet. They were kept full time on large pastures with run-in-sheds, free access to water, mineral salt blocks, and were grazing in the summer and fed grass hay in the winter. All horses studied had either naturally occurring Cul hypersensitivity or were clinically healthy. Allergic horses (n=7) included 5 mares (11-17 years, median 16 years), 1 gelding (age 9) and 1 stallion (age 8). Healthy horses (n=6) included 4 mares (7-9 years, median 8 years) and 2 geldings (age 8). For CD23+ cell phenotyping experiments, 3 allergic mares, 1 allergic gelding, 1 stallion, and 4 healthy mares were used. For FACS sorting and PCR analysis 2 allergic mares and 1 allergic gelding were used. For longitudinal analysis of ex vivo peripheral blood mononuclear cells (PBMC), all 7 allergic and 6 healthy horses were compared at each timepoint except for March, which included 7 allergic and 4 healthy horses.
All horses were given a clinical allergy score every 2-4 weeks for the duration of the study. Scores were assigned based on pruritis (0-3), alopecia (0-4), and dermatitis (0-3) and total scores ≥3 were considered allergic. All allergic horses displayed allergic signs with scores above 3 for at least one year before, and for the duration of this study (Table 2). Cul-specific hypersensitivity was further confirmed by intradermal skin testing with Cul whole body extract (WBE; Stallergenes Greer Inc., Cambridge, MA, USA) in comparison to saline and histamine controls. Allergic horses developed an immediate skin reaction to Cul WBE. Nonallergic horses never exhibited clinical allergy or scoring above 3 and did not react to intradermal Cul injections.
For extracellular staining and analysis, PBMC or PBL were incubated for 15 min with different antibody master mixes (Table 3) in PBS-BSA (0.5% (w/v) BSA, 0.02% (w/v) NaN3, all from Sigma-Aldrich, St. Louis, MO, USA). Monoclonal antibodies (mAbs) against different horse cell surface markers or Ig isotypes were conjugated to Alexa fluorochrome 647, Alexa fluorochrome 488, or biotin according to manufacturer's protocols (Thermo Fisher Scientific, Waltham, MA, USA). Master mixes 1-10 (Table 3) were used to phenotype the different CD23+ populations in equine PBMC. Master mixes 11-15 were used to measure intracellular IgE production. Master mix 16 was used for cell sorting of CD23hi/IgE+ cells and IgE− B cells. Master mix 17 was used to measure IgE+ plasmablasts in allergic and healthy horses at different timepoints. Streptavidin-PE (Jackson ImmunoResearch Laboratories) was used to label biotinylated mAbs.
For intracellular staining and analysis, fixed PBMC were incubated first with master mix 11 (Table 3) in PBS-BSA to label cell surface IgE and CD23. Cells were subsequently incubated with streptavidin-PE to label biotinylated CD23. Cells were then washed once in saponin buffer (0.5% Saponin 0.5% BSA 0.02% NaN3 in PBS, all from Sigma-Aldrich, St. Louis, MO, USA), and incubated with master mix 12 in saponin buffer to label intracellular IgE. As a control, one aliquot of cells was incubated with master mix 13, in PBS-BSA instead of saponin buffer. Isotype controls were included in master mixes 14 and 15, which were subsequently stained on an additional PBMC aliquot to set the IgE+ gates.
Samples were recorded on a BD FACS Canto II flow cytometer and data analysis was performed with FlowJo version 10.4 (FlowJo, Ashland, OR, USA). A total of 100,000 events/sample were recorded for all master mixes except mixes 2 and 3, which recorded 50,000 events/sample. All flow cytometry images were gated first to exclude doublets and second to gate on PBMC by forward and side scatter characteristics. IgE+ cells were analyzed quantitatively.
Following PBMC isolation and removal of surface-bound IgE, 1×107 cells were incubated with mAb master mix 16 (Table 3), including a viability marker (Thermo Fisher Scientific, Waltham, MA, USA), to isolate IgE+ plasmablasts by FACS. IgE mAb 176 was used for cell sorting due to its inability to induce crosslinking of receptor-bound IgE. All cell sorting was performed at 4° C. to minimize activation of B cells. Cells were sorted on a BD FACS Aria Fusion Sorter at the Cornell Institute of Biotechnology's flow cytometry core facility. Sorting was performed through a 100 μm nozzle at 20 psi in a sterile hood. Compensation was calculated with input from single stained UltraComp beads and amine reactive beads (Thermo Fisher Scientific, Waltham, MA, USA). Live CD23hi/IgE+ cells were collected into cell culture medium (DMEM supplemented with 1% (v/v) non-essential amino acids, 2 mM L-glutamine, 50 μM 2-mercaptoethanol, 50 μg/ml gentamicin, 100 U/ml penicillin, 100 μg/ml streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) and 10% FCS (Atlanta biological, Flowery Branch, GA, USA)) at 4° C. Live CD23lo/IgE− cells were simultaneously sorted and collected for comparison. Sorted fractions were counted and tested for viability using trypan blue exclusion. All samples were >88% viable (average 93% live cells). To confirm morphology of sorted fractions, cytospin smears were prepared in a cytocentrifuge (Thermo Shandon Cytospin 4, Thermo Fisher Scientific, Waltham, MA, USA) at 176 g for 4 min at room temperature, and then stained with Wright's stain. Morphology descriptions were performed by a single observer who was blinded to the origin of the cells and the sorting procedure.
Sorted CD23hi/IgE+ cells and IgE− B cells (1×104 cells each) from three allergic horses in October were frozen in 3:1 Trizol LS:cell culture medium (Thermo Fisher Scientific, Waltham, MA, USA) at −80° C. until processed for RNA extraction. RNA was extracted in the aqueous layer with chloroform (Thermo Fisher Scientific, Waltham, MA, USA) in Phaselock Gel Heavy tubes (VWR International, Radnor, PA, USA). RNA pellets were co-precipitated with GlycoBlue (Ambion Incorporated, Austin, TX, USA) and washed once with isopropanol and three times with ethanol. Pellets were air dried and resuspended in 20 μl nuclease-free water (GrowCells, Irvine, CA, USA). RNA concentration and quality were measured via nanodrop. Equal amounts of extracted RNA were converted to cDNA using SuperScript™ VILO™ Reverse Transcriptase (RT) and no-Reverse Transcriptase control (no-RT) master mixes (Thermo Fisher Scientific, Waltham, MA, USA).
Primers specific for the equine IgE heavy chain constant region (IGHE) were synthesized by Integrated DNA Technology (Coralville, IA, USA). The transcript was amplified from 0.8 μl cDNA from FACS sorted CD23hi/IgE+ cells, with 500 nM primers (Table 1, Forward primer: GTCTCCAAGCAAGCCCCATTA (SEQ ID NO: 21); reverse primer: TCGCAAGCTTTACCAGGGTCTTTGGACACCTC (SEQ ID NO: 22)) and Platinum™ Superfi PCR master mix (Invitrogen, Carlsbad, CA, USA). The PCR reaction was run with an initial denaturing of 94° C. for 10 seconds, then 30 cycles of 94° C. 10 sec, 57° C. 10 sec and 72° C. 30 sec, followed by a final elongation step at 72° C. 5 min and cooled to 4° C. PCR products were extracted from a 1% agarose gel (Invitrogen, Carlsbad, CA, USA), cloned into a pCR4-TOPO plasmid (Invitrogen, Carlsbad, CA, USA) and sequenced by Illumina Sequencing.
Additional gene specific primers (Table 1) were designed to span intron-exon boundaries with Integrated DNA Technology's PrimerQuest tool and were synthesized by Integrated DNA Technology (Coralville, IA, USA). Primers were also aligned with the equine genome and did not have any predicted off-target amplicons of similar product size. Therefore, to confirm primer specificity, PCR product size was confirmed on a 2% agarose gel (Invitrogen, Carlsbad, CA, USA) with a 10 bp ladder (Thermo Fisher Scientific, Waltham, MA, USA). Beta actin (ACTB) was used as a reference gene.
Quantitative real-time PCR (qPCR) was performed with 10 μl reactions each containing a cDNA equivalent obtained from a reverse transcriptase reaction of 3.4 ng mRNA, 500 nM forward and reverse primers and SsoAdvanced Universal SYBR green master mix (Bio-Rad Laboratories, Raleigh, NC, USA). No-RT controls were run simultaneously for all samples and primer pairs. The qPCR reaction was run on a QuantStudio 5 thermocycler (Thermo Fisher Scientific, Waltham, MA, USA) with an initial dissociation at 95° C. for 30 s, then 40 cycles of 95 C.° for 15 s and 60° C. for 15 s, followed by melt curve analysis. For qPCR results, delta Ct (dCt) values were calculated for all samples where dCt=Ct[target gene]−Ct[reference gene]. dCt values were calculated using both reference genes, ACTB or B2M. In general, the smaller the dCt, the greater the amount of mRNA in the sample. The mean difference in gene expression (ddCt) was calculated where ddCt is the average of dCt[IgE− B cells]−dCt[CD23hi/IgE+ cells] from each individual. Positive ddCt values represent increased gene expression in CD23hi/IgE+ cells compared to IgE− B cells.
D'Agostino and Pearson tests were performed on all data sets and confirmed that the data were not normally distributed. Thus, non-parametric tests were used for data analysis. To compare IgE+ plasmablast frequencies between allergic and healthy horses at different timepoints, a Holm-Sidak multiple comparisons test was used. A nonparametric Spearman rank correlation was calculated for all horses to compare each individual's clinical allergy score or concentration of secreted IgE to the peripheral IgE+ plasmablast percentage. A Mann-Whitney test was used to compare secreted IgE concentrations between allergic and healthy groups. A paired t test was used to compare dCt values between sorted CD23hi/IgE+ cells and IgE− B cells. Gene expression graphs plot individual and average dCt values for each gene and cell fraction. A paired t test was also used to compare IgE secretion between IgE− B cells and IgE+ plasmablast FACS sorted samples. qPCR gene expression and IgE secretion were considered normally distributed due to the comparison of two cell types within each individual. All graphs plot median and range unless specified otherwise and p values <0.05 were considered significant. Analysis was performed with GraphPad Prism software version 8 (GraphPad Software Inc., La Jolla, CA, USA).
This application claims the benefit of U.S. Provisional Patent Application No. 63/255,756, filed Oct. 14, 2021, the contents of which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/078106 | 10/14/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63255756 | Oct 2021 | US |
