Bystander T cells in human immune responses to dengue antigens

Bystander activation of T cells refers to activation of T cells specific for an antigen X during an immune response against antigen Y. Such an activation of T cells is independent of TCR signaling and occurs through cytokines by novel activating receptors. It has been reported in models of viral infections such as herpes simplex virus, LCMV and HIV leading to proliferation of memory T cells and subsequent production of cytokines. Studies have also found bystander CD8+ T cell activation in response to intracellular bacteria. Dengue viral infection is the cause of dengue fever and dengue hemorrhagic fever. Previous studies have noted that in response to dengue infection, many pro-inflammatory cytokines are released during acute infection. Thus, bystander T cell activation may possibly occur during dengue infection.

This study was conducted by the lab of Lertmemongkolchai, The Center for Research and Development of Medical Diagnostic Laboratories, Thailand. The investigators aimed to investigate the activation of bystander T cell activation in healthy children living in endemic areas who might be vulnerable re-infection with dengue virus. Thus, they examined IFN-gamma production, which is the established indicator for bystander T cell activation, after restimulating with inactivated dengue viral antigen in vitro. Furthermore, the authors looked for bystander T cell activity by resistance to cyclosporine S (CsA), as CsA is known to inhibit T cell activation by via the TCR dependent pathway.

Source: Suwannasaen D, Romphruk A, Leelayuwat C, Lertmemongkolchai G. Bystander T

cells in human immune responses to dengue antigens. BMC Immunol. 2010 Sep

20;11:47. PubMed PMID: 20854672; PubMed Central PMCID: PMC2949776.

 

The major results of the study are as follows:

1. Healthy Thai school children could produce IFN-gamma in response to inactivated dengue virus serotype 2 in vitro: The investigator took blood samples from 55 healthy Thai school children and co-cultured them with control (medium alone), stimulators including Phytohemagglutinin (PHA) and combination of cytonies, IL12 and IL15, or Den2 in the presence or absence of CsA for 48 hrs. Cyclosporin A (CsA) is known to inhibit T cell activation via the TCR –dependent pathway. The culture supernatants were then assayed for IFN-gamma by sandwich ELISA. In the presence of positive control stimulators, a higher production of IFN-gamma was observed in compared to negative controls. In the presence of CsA, the production of IFN-gamma was inhibited. This inhibition was observed in case of stimulation by PHA and not with IL-15, IL-12. These results indicate that CsA sufficiently inhibit IFN-gamma production via the TCR-dependent but not the cytokine dependent pathway. The authors also observed that inhibition by CsA in response to Den2 and PHA treatment was very high in comparison to IL-12, IL-15 treatment indicating that IFN-gamma production by Den2 was mainly through TCR dependent pathway. To validate whether CsA does inhibit all TCR-stimulated IFN-gamma activation, the authors used a MHC class I restricted T cell epitope control of pooled peptides (CEF) of cytomegalovirus, EBV and influenza virus. The authors used five whole blood samples from healthy school children and cultured them with medium control, Den2, PHA, and CEF in the presence of absence of CsA. The results showed that CsA completely inhibited IFN-gamma production in response to CEF-stimulation but it only partially inhibits IFN-gamma production in response to stimulation by Den2. These results showed that CsA could inhibit all IFN-gamma production from TCR-dependent activation.
   
2. Identification of IFN-gamma+ cells that respond to Den2: Eighteen children who showed high IFN-gamma production were followed up to determine the IFN-gamma producing cells. Thus, the investigators co-cultured whole blood samples with medium and 18 HA units Den2 in the presence or absence of CsA. These cells were then analyzed by flow cytometry. The types of IFN-gamma+ cells were then analyzed by the combination of tri-CD3, FITC-CD8 and PE-CD4 or PE-CD56 and compared in the presence or absence of CsA. The results showed that percentage of IFN-gamma + cells in response to stimulation with Den2 was significantly higher than that in response to control (medium alone). In the presence of CsA, there was a change in IFN-gamma+ cells, which was due to decrease in number of NK cells and increase in number of T cells. The results suggested that dengue virus causes bystander T cell activation in vitro as some CD4+ and CD8+ T cells resisted the effect of CsA. The IFN-gamma + cells consisted of NK cells, CD4+ T cells and CD8+ T cells. The distribution of bystander or CsA-resistant CD4+ ranged from 10-59% and distribution of bystander or CD8+ cells ranged from 7-39%.
   
3. Kinetics of bystander T and NK cell activation: The authors next sought to describe characteristics of activated bystander T cells. Thus, the authors compared kinetics of IFN-gamma production by bystander T cells with those of NK cells and activated specific T cells. The authors studied blood samples from seven school children after 12h, 24h, 36h stimulation. The IFN-gamma+ cells were detected as early as 12h post stimulation. All the seven samples showed similar kinetics of bystander CD4+, CD8+ and NK cells. After 12h post stimulation, bystander CD4+/CD8+ cells were detected while specific CD4+/CD8+ cells were not detected. In contrast, after 24 h post stimulation the proportion of bystander and specific CD4/CD8 cells were equal.
   
4. IL-12 dependent pathway mediated T cell to produce IFN-gamma- The authors next treated whole blood samples from healthy donors with anti-cytokine antibodies, anti-IL-12, anti-IL-15 and anti-IL-18 and then treated these samples with heat –inactivated Burkholderia pseudomallei, which is a strong bystander or cytokine-dependent T cell inducer. After 48 h, culture supernatants were analyzed for IFN-gamma by ELISA. The results showed that all these three anti-cytokine antibodies could decrease IFN-gamma production by B. pseudomallei. To further investigate the production of IFN-gamma in response to dengue virus, the authors took eleven blood samples from healthy adult blood donors, treated with anti-cytokine antibodies against IL-12, IL-15 and IL-18 and then tested IFN-gamma production from culture supernatants. Anti-IL12 antibody but not anti-IL15 or anti-IL-18 antibody caused a statistically significant reduction in IFN-gamma production in response to dengue virus stimulation. The results suggest that IFN-gamma is mainly activated via IL-12 dependent pathways. When CsA was added in addition to anti-IL12, IFN-gamma production was completely inhibited. The authors then selected three representative blood samples to characterize the intracellular source of IFN-gamma by flowcytometry. They observed that major IFN-gamma producing cells were NK, CD4+ and CD8+ T cells. The authors next selected two representative samples to determine the effect of neutralizing antibodies on IFN-gamma production. Results showed that anti-IL12 and CsA cause a decrease in IFN-gamma producing cells derived from both CD4 and CD8 cells. The results also showed that CD4+ T cells were equally sensitive to anti-IL-12 and CsA, while CD8+ T cells were less sensitive to anti-IL12 than CsA. These results confirmed that bystander T cells are produced in response to dengue virus and these T cells produce IFN-gamma via IL-12 dependent pathway.