| 1. |
- Jahnmatz, Peter, et al.
(author)
-
An antigen-specific, four-color, B-cell FluoroSpot assay utilizing tagged antigens for detection
- 2016
-
In: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 433, s. 23-30
-
Journal article (peer-reviewed)abstract
- The FluoroSpot assay, a variant of ELISpot utilizing fluorescent detection, has so far been used primarily for assessment of T cells, where simultaneous detection of several cytokines has allowed a more qualitative analysis of functionally distinct T cells. The potential to measure multiple analytes also presents several advantages when analyzing B cells. Our aim was to develop a B-cell FluoroSpot assay adaptable to studies of a variety of antigens. The assay utilizes anti-IgG antibodies immobilized in 96-well filter membrane plates. During cell culture, IgG antibodies secreted by antibody-secreting cells (ASCs) are captured in the vicinity of each of these cells and the specificity of single ASCs is defined using antigens for detection. The antigens were labeled with biotin or peptide tags enabling secondary detection with fluorophore-conjugated streptavidin or tag-specific antibodies. The assay, utilizing up to four different tag systems and fluorophores simultaneously, was evaluated using hybridomas and immunized splenocytes as ASCs. Assay variants were developed that could: i) identify multiple ASCs with different antigen specificities; ii) detect ASCs showing cross-reactivity with different but related antigens; and iii) define the antigen-specificity and, by including anti-IgG subclass detection reagents, simultaneously determine the IgG subclass of antibodies secreted by ASCs. As demonstrated here, the B-cell FluoroSpot assay using tag-based detection systems provides a versatile and powerful tool to investigate antibody responses by individual cells that can be readily adapted to studies of a variety of antigen-specific ASCs.
|
|
| 2. |
- Jahnmatz, Peter, et al.
(author)
-
Memory B-Cell Responses Against Merozoite Antigens After Acute Plasmodium falciparum Malaria, Assessed Over One Year Using a Novel Multiplexed FluoroSpot Assay
- 2021
-
In: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 11
-
Journal article (peer-reviewed)abstract
- Memory B cells (MBCs) are believed to be important for the maintenance of immunity to malaria, and these cells need to be explored in the context of different parasite antigens and their breadth and kinetics after natural infections. However, frequencies of antigen-specific MBCs are low in peripheral blood, limiting the number of antigens that can be studied, especially when small blood volumes are available. Here, we developed a multiplexed reversed B-cell FluoroSpot assay capable of simultaneously detecting MBCs specific for the four Plasmodium falciparum blood-stage antigens, MSP-1(19), MSP-2, MSP-3 and AMA-1. We used the assay to study the kinetics of the MBC response after an acute episode of malaria and up to one year following treatment in travelers returning to Sweden from sub-Saharan Africa. We show that the FluoroSpot assay can detect MBCs to all four merozoite antigens in the same well, and that the breadth and kinetics varied between individuals. We further found that individuals experiencing a primary infection could mount and maintain parasite-specific MBCs to a similar extent as previously exposed adults, already after a single infection. We conclude that the multiplexed B-cell FluoroSpot is a powerful tool for assessing antigen-specific MBC responses to several antigens simultaneously, and that the kinetics of MBC responses against merozoite surface antigens differ over the course of one year. These findings contribute to the understanding of acquisition and maintenance of immune responses to malaria.
|
|
| 3. |
- Jahnmatz, Peter
(author)
-
Methods for studying memory B-cell immunity against malaria
- 2020
-
Doctoral thesis (other academic/artistic)abstract
- Plasmodium falciparum malaria remains one of the world’s deadliest infectious diseases and the search for an effective vaccine is highly warranted. Memory B cells (MBCs) and the antibodies they produce, once activated, is believed to play an important role in the protective immunity against malaria, but the mechanism of acquiring and maintaining these cells is poorly understood. New and sensitive tools able of gathering detailed information regarding the development and maintenance of antigen-specific MBCs could increase the understanding of protective immunity but also be used for the evaluation of new vaccines. In Study I, we developed the reversed B-cell FluoroSpot assay, a new assay format based on an established technique for single-cell analysis. Using hybridomas and splenocytes from immunized mice together with a tag/anti-tag approach for detection, we showed proof-of-principle that the assay could be used for multiplex analysis of single B cells specific to four different antigens simultaneously, as well as detecting B cells displaying cross-reactivity against antigen variants. In Study II, we adapted the assay for studies on humans and measured MBC responses against hepatitis B virus, tetanus toxoid and cytomegalovirus. We also measured MBC frequencies before and after vaccination against hepatitis B and used new FluoroSpot reader functions to assess spot volume. We showed that the assay could be used to detect B cells against all of the antigens simultaneously and also changes in MBC frequencies and spot volume before and after vaccination. In Study III, we adapted the multiplex assay further for studies on P. falciparum antigen-specific MBCs and used it to study the kinetics of MBC responses in primary infected and previously exposed travelers diagnosed with malaria in Sweden. We showed that primary infected individuals could acquire and maintain P. falciparum-antigen specific MBCs as efficiently as previously exposed individuals during a one year follow up period, but that the maintenance and magnitude of antibody levels in plasma were higher in the previously exposed individuals. In Study IV, we used the assay developed in Study III to analyze P. falciparum antigen-specific MBCs in children living in areas with endemic transmission of malaria in Kenya. We identified that high levels of MBCs against certain P. falciparum antigens were associated with a reduced risk of a subsequent clinical malaria episode, and that proportions of MBCs specific to some, but not all, P. falciparum antigens, increase with age, but also some decrease with cumulative number of infections. We conclude that the multiplex FluoroSpot method developed in this thesis provide insights towards the acquisition and maintenance of P. falciparum malaria-induced MBCs. We believe that the reversed B-cell FluoroSpot assay is a sensitive and highly adaptable method to assess MBC responses against multiple antigens and will be a powerful tool for future studies on protective immunity to malaria, but also other fields of research.
|
|
| 4. |
- Jahnmatz, Peter, et al.
(author)
-
Multiplex analysis of antigen-specific memory B cells in humans using reversed B-cell FluoroSpot
- 2020
-
In: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 478
-
Journal article (peer-reviewed)abstract
- Analysis of B-cell specificities at the single cell level provides important information on how the B-cell compartment responds when challenged by infection or vaccination. We recently developed a reversed B-cell FluoroSpot assay and showed that it could be used to detect B cells specific for different antigens simultaneously in a mouse model. The aim of this study was to further develop the method to detect and quantify antigen-specific memory B cells (MBCs) in humans where circulating antigen-specific cells are less frequent. We show that MBCs specific for three antigens, tetanus toxoid, hepatitis B surface antigen and cytomegalovirus pp65, could be detected simultaneously in one well. In addition to enumerating antigen-specific MBCs, we also assessed the spot volume to estimate the intensity of the response in individual cells and found this to be a new and sensitive approach to study MBC responses after vaccination. This unique B-cell FluoroSpot approach provides a simple and sensitive multiplex analysis of MBCs and can be adapted to most antigens and host species.
|
|
| 5. |
- Jahnmatz, Peter, et al.
(author)
-
Plasmodium falciparum-Specific Memory B-Cell and Antibody Responses Are Associated With Immunity in Children Living in an Endemic Area of Kenya
- 2022
-
In: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 13
-
Journal article (peer-reviewed)abstract
- Identifying the mechanism of naturally acquired immunity against Plasmodium falciparum malaria could contribute to the design of effective malaria vaccines. Using a recently developed multiplexed FluoroSpot assay, we assessed cross-sectional pre-existing memory B-cells (MBCs) and antibody responses against six well known P. falciparum antigens (MSP-119, MSP-2 (3D7), MSP-2 (FC27), MSP-3, AMA-1 and CSP) and measured their associations with previous infections and time to clinical malaria in the ensuing malaria season in Kenyan children. These children were under active weekly surveillance for malaria as part of a long-term longitudinal malaria immunology cohort study, where they are recruited from birth. After performing Cox regression analysis, we found that children with a breadth of three or more antigen-specific MBC or antibody responses at the baseline had a reduced risk for malaria in the ensuing P. falciparum transmission season. Specifically, MBC responses against AMA-1, MSP-2 (3D7) and MSP-3, as well as antibody responses to MSP-2 (3D7) and MSP-3 were prospectively associated with a reduced risk for malaria. The magnitude or breadth of MBC responses were however not correlated with the cumulative number of malaria episodes since birth. We conclude that increased breadth for merozoite antigen-specific MBC and antibody responses is associated with protection against malaria.
|
|
| 6. |
- Sundling, Christopher, et al.
(author)
-
B cell profiling in malaria reveals expansion and remodeling of CD11c+ B cell subsets
- 2019
-
In: JCI Insight. - : American Society for Clinical Investigation. - 2379-3708. ; 4:9
-
Journal article (peer-reviewed)abstract
- Humoral immunity is important in limiting clinical disease in malaria, yet the longitudinal B cell response to infection remains unclear. We performed a 1-year prospective study in patients treated for acute Plasmodium fakiporum malaria for the first time or with previous exposure to the disease. Using an unbiased exploratory approach with mass cytometry, followed by targeted flow cytometry, we found that approximately 80% of mature B cells that proliferated in response to acute infection expressed CD11c. Only approximately 40% of CD11c+ B cells displayed an atypical B cell phenotype, with the remaining cells primarily made up of activated and resting memory B cells. The CD11c+ B cells expanded rapidly following infection, with previous exposure to malaria resulting in a significantly larger increase compared with individuals with primary infection. This was attributed to an expansion of switched CD11c+ B cells that was absent in primary infected individuals. The rate of contraction of the CD11c+ B cell compartment was independent of previous exposure to malaria and displayed a slow decay, with a half-life of approximately 300 days. Collectively, these results identify CD11c as a marker of B cells responding to malaria and further highlight differences in primary and secondary B cell responses during infection.
|
|
| 7. |
- Tiselius, Eva, et al.
(author)
-
Bone Marrow-Suppressive Treatment in Children Is Associated with Diminished IFN-γ Response from T Cells upon Polyclonal and Varicella Zoster Virus Peptide Stimulation
- 2024
-
In: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 25:13
-
Journal article (peer-reviewed)abstract
- Severe haematological diseases and lymphoid malignancies require bone marrow (BM)-suppressive treatments. Knowledge regarding the impact of BM-suppressive treatments on children's memory T cells is very limited. Memory T cells play a crucial role in defending against herpesviruses, which is particularly relevant in paediatric cancer care. We studied 53 children in total; 34 with cancer and 2 with severe haematological disorders, with some receiving BM-suppressive treatment with or without allogeneic-haematopoietic stem cell transplantation (allo-HSCT), alongside 17 healthy controls. We focused on peripheral blood proportions of memory T-cell subsets using flow cytometry and analysed cytokine-secreting T cells with a four-parameter FluoroSpot assay in response to T-cell mitogen and varicella zoster virus (VZV) peptides. Patients on BM-suppressive treatment showed increased clusters of differentiation (CD)4+ and CD8+ effector memory (TEM)/terminally differentiated effector (TEFF) T cells compared to the healthy controls. They also exhibited, amongst other things, when compared to the healthy controls, a reduced total number of cytokine-secreting cells, by means of interferon (IFN)-γ, interleukin (IL)-17A, IL-10, and IL-22, following mitogen activation. A diminished IFN-γ response among the children with BM-suppressive treatment was observed upon VZV-peptide stimulation, compared to the healthy children. Collectively, the findings herein indicate that the children who are undergoing or have finished BM-suppressive treatment display qualitative differences in their T-cell memory compartment, potentially increasing their susceptibility to severe viral infections and impacting their immunotherapy, which relies on the functional ability of autologous T cells.
|
|
