Abstract
Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, continues to cause significant morbidity and mortality world-wide. CD8+ T cells are an important component of the cell mediated immune (CMI) response against S. Typhi. Recently, interleukin (IL)-17A has been shown to contribute to mucosal immunity and protection against intracellular pathogens. To investigate multifunctional IL-17A responses against S. Typhi antigens in T memory subsets, we developed multiparametric flow cytometry methods to detect up to 6 cytokines/chemokines (IL-10, IL-17A, IL-2, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-1β (MIP-1β)) simultaneously. Five volunteers were immunized with a 4 dose regimen of live-attenuated S. Typhi vaccine (Ty21a), peripheral blood mononuclear cells (PBMC) were isolated before and at 11 time points after immunization, and CMI responses were evaluated. Of the 5 immunized volunteers studied, 3 produced detectable CD8+ T cell responses following stimulation with S. Typhi-infected autologous B lymphoblastoid cell lines (B-LCL). Additionally, 2 volunteers had detectable levels of intracellular cytokines in response to stimulation with S. Typhi-infected HLA-E restricted cells. Although the kinetics of the responses differed among volunteers, all of the responses were bi- or tri-phasic and included multifunctional CD8+ T cells. Virtually all of the IL-17A detected was derived from multifunctional CD8+ T cells. The presence of these multifunctional IL-17A+ CD8+ T cells was confirmed using an unsupervised analysis program, flow cytometry clustering without K (FLOCK). This is the first report of IL-17A production in response to S. Typhi in humans, indicating the presence of a Tc17 response which may be important in protection. The presence of IL-17A in multifunctional cells co-producing Tc1 cytokines (IL-2, IFN-γ and TNF-α) may also indicate that the distinction between Tc17 and Tc1 responses in humans is not as clearly delineated as suggested by in vitro experiments and animal models.
Highlights
Given the prominent role that multifunctional T cells have been shown to have in the host defense from other human infections [29,30,31], we investigated the concomitant production of IL-17A with 5 other cytokines/ chemokines (IL-10, IL-2, IFN-c, tumor necrosis factor-a (TNF-a), and macrophage inflammatory protein-1b (MIP-1b) as a first step to investigate the potential role of multifunctional T cells in protection from S
peripheral blood mononuclear cells (PBMC) were stained with monoclonal antibodies against CD3, CD4, CD8, CD14, CD19, CD45RA, and CD62L followed by intracellular staining for CD69, IL-10, IL-17A, IL-2, IFN-c, TNF-a, and MIP1b
CD3+ CD8+ T cells were categorized by their expression of CD62L and CD45RA into naıve T cells (TN; CD62L+ CD45RA+), T central memory cells (TCM; CD62L+ CD45RA-), T effector memory (TEM; CD62LCD45RA-), and T effector memory CD45RA+ (TEMRA; CD62LCD45RA+) as previously described (Figure S1) [34]
Summary
Typhi), a human restricted pathogen, is the causative agent of typhoid fever. S. Typhi is a Gram-negative, facultative intracellular pathogen that poses a significant threat to public health, in the developing world [1,2]. Typhoid fever is responsible for an estimated 21 million illnesses and 200,000 deaths annually and increasing antibiotic resistance among S. Typhi infected cells [10,18]. Multifunctional CD8+ T cells have been identified in volunteers immunized with live-attenuated typhoid vaccine Ty21a [13]. The latter study has shown multiphasic cytokine production by CD8+ T cells in response to human leukocyte antigen E (HLA-E)-restricted antigenic stimulation [13]
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