Four-Parameter FluoroSpot Assay Reveals That the Varicella Zoster Virus Elicits a Robust Memory T Cell IL-10 Response throughout Childhood.

During childhood, the composition and function of the T cell compartment undergoes significant changes. In healthy individuals, primary infection with herpesviruses is followed by latency, and occasional subclinical reactivation ensures transmission and contributes to an emerging pool of memory T cells. In immunocompromised individuals, herpesviruses can be life threatening. However, knowledge about the spectrum of virus-specific cytokine responses is limited. Here, we investigated peripheral blood mononuclear cells (PBMCs) from children with differential carrier statuses for cytomegalovirus (CMV), Epstein-Barr virus (EBV), and varicella zoster virus (VZV) (n = 32, age 1 to 17 years). We examined memory T cell subsets as well as IFN-γ-, IL-10-, IL-17A-, and IL-22-producing T cells after polyclonal activation or stimulation with viral peptides using flow cytometry and a 4-parameter FluoroSpot assay. Age and herpesvirus carriage influenced the size of the memory T cell subsets. A positive association between age and the number of IFN-γ-, IL-17A- and IL-22-producing T cells was found following polyclonal activation. For CMV, age was positively associated with IL-17A spot-forming cells (SFC), while for VZV, age was negatively associated with IL-22 and positively associated with IFN-γ SFC. Upon activation with CMV, VZV, and EBV peptides, IFN-γ SFCs dominated. Notably, VZV responses were characterized by a higher IL-10 SFC population compared to both CMV and EBV. Our findings suggest that cytokine responses vary across herpesvirus-type-specific memory T cells and may more adequately reflect their composition. An observed deviation between polyclonal and herpesvirus-specific T cell cytokine responses in children needs to be considered when interpreting the associations between herpesvirus carrier status and bulk T cell reactivity. In summary, these findings may have implications for the treatment of immunocompromised patients. IMPORTANCE Infection with herpesviruses accounts for 35 to 40 billion human cases worldwide. Despite this, little is known about how herpesviruses shape the immune system in the asymptomatic carrier. Particularly in children, primary infection is connected to no or mild symptoms ahead of latency for life. Most research on cellular responses against herpesviruses focuses on inflammatory cytokines associated with antiproliferative and antitumor mechanisms and not the spectrum of cytokine responses in healthy humans. This study investigated four divergent cytokine-producing T cell responses to herpesviruses, reflecting different immunological functions. Three common childhood herpesviruses were selected: Epstein-Barr virus, cytomegalovirus, and varicella-zoster virus. Curiously, not all viruses induced the same pattern of cytokines. Varicella-zoster responses were characterized by IL-10, which is considered regulatory. Besides broadening understanding of responses to herpesviruses, our results raise the possibility that reactivation of varicella-zoster may be counterproductive in cancer treatment through the action of IL-10-producing T-cells.

Plasmodium falciparum-Specific Memory B-Cell and Antibody Responses Are Associated With Immunity in Children Living in an Endemic Area of Kenya.

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.

Memory B-Cell Responses Against Merozoite Antigens After Acute Plasmodium falciparum Malaria, Assessed Over One Year Using a Novel Multiplexed FluoroSpot Assay.

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-119, 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.

Multiplex analysis of antigen-specific memory B cells in humans using reversed B-cell FluoroSpot.

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.

B cell profiling in malaria reveals expansion and remodelling of CD11c+ B cell subsets.

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 P. falciparum 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 ~80% of mature B cells that proliferated in response to acute infection expressed CD11c. Only ~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 to 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 ~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.

Detection of Cross-Reactive B Cells Using the FluoroSpot Assay.

B cell ELISpot enables a sensitive analysis of antigen-specific B cells at the single cell level but is limited to the analysis of reactivity with a single antigen. By reversing the B cell ELISpot and using anti-IgG capture antibodies instead of coated antigen, the specificity of antibodies secreted by B cells can be defined using soluble tagged antigen for detection. When combining this approach with fluorescent detection of the antigen in a B cell FluoroSpot assay, reactivity with multiple antigens can be defined. In the protocol described herein, splenocytes from a mouse immunized with an antigen were analyzed for their reactivity with the antigen used for immunization and for cross-reactivity with a different but structurally related antigen. Using this assay, we found that at least 15% of the B cells displayed detectable cross-reactivity. B cell FluoroSpot utilizing multiple antigens provides a tool for a single-cell analysis of B cell cross-reactivity, for example, with variable and polymorphic antigens found in various pathogens; or analysis of other types of immune responses where analysis of cross-reactivity is of interest. It is also possible to simultaneously analyze B cell reactivity to completely different antigens.

An antigen-specific, four-color, B-cell FluoroSpot assay utilizing tagged antigens for detection.

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.