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High pro-inflammatory cytokine secretion and loss of high avidity cross-reactive cytotoxic T-cells during the course of secondary dengue virus infection - PubMed

  • ️Mon Jan 01 2007

High pro-inflammatory cytokine secretion and loss of high avidity cross-reactive cytotoxic T-cells during the course of secondary dengue virus infection

Tao Dong et al. PLoS One. 2007.

Abstract

Background: Dengue is one of the most important human diseases transmitted by an arthropod vector and the incidence of dengue virus infection has been increasing - over half the world's population now live in areas at risk of infection. Most infections are asymptomatic, but a subset of patients experience a potentially fatal shock syndrome characterised by plasma leakage. Severe forms of dengue are epidemiologically associated with repeated infection by more than one of the four dengue virus serotypes. Generally attributed to the phenomenon of antibody-dependent enhancement, recent observations indicate that T-cells may also influence disease phenotype.

Methods and findings: Virus-specific cytotoxic T lymphocytes (CTL) showing high level cross reactivity between dengue serotypes could be expanded from blood samples taken during the acute phase of secondary dengue infection. These could not be detected in convalescence when only CTL populations demonstrating significant serotype specificity were identified. Dengue cross-reactive CTL clones derived from these patients were of higher avidity than serotype-specific clones and produced much higher levels of both type 1 and certain type 2 cytokines, many previously implicated in dengue pathogenesis.

Conclusion: Dengue serotype cross-reactive CTL clones showing high avidity for antigen produce higher levels of inflammatory cytokines than serotype-specific clones. That such cells cannot be expanded from convalescent samples suggests that they may be depleted, perhaps as a consequence of activation-induced cell death. Such high avidity cross-reactive memory CTL may produce inflammatory cytokines during the course of secondary infection, contributing to the pathogenesis of vascular leak. These cells appear to be subsequently deleted leaving a more serotype-specific memory CTL pool. Further studies are needed to relate these cellular observations to disease phenotype in a large group of patients. If confirmed they have significant implications for understanding the role of virus-specific CTL in pathogenesis of dengue disease.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. (a) Ex vivo pD2 and pD3/4 tetramer staining of PBMC from patient BC307.

Freshly thawed cryopreserved PBMCs from BC307 were stained with A11 pD3/4 and pD2 tetramers and CD8 antibody. The plots are gated on CD8 positive lymphocytes. The majority of cells in the acute sample (left) are specific for pD3/4 but there is a significant population cross-reactive with this and pD2. The convalescent sample (right) shows only pD3/4 specific cells. (b,c,d) Highly cross-reactive T cells can be expanded from acute but not convalescent PBMC from dengue patients. Short term CTL lines were generated by pulsing PBMC with 2 µM of pD2 and stained with pD2 and pD3/4 tetramers on either the 14th (BC307) or 20th (MD856, MD893) day after stimulation. The highly cross-reactive populations apparent in the acute sample are not detectable in the convalescent sample taken one month later when more serotype specific populations have appeared.

Figure 2
Figure 2. Highly cross-reactive CTL are more activated than partially cross-reactive CTL.

Frozen PBMC from patient BC307 on day 21 of illness were stained with pD1 and pD3/4 tetramers together with CD38 and CD8. (a) CD38 staining of the whole CD8+ population acutely and at day 21. (b) Lymphocytes gated on CD8+ cells and co-stained with pD1 and pD3/4 tetramers. Highly cross-reactive cells (R7) show higher levels of expression of CD38 (d) than partially cross-reactive cells (c).

Figure 3
Figure 3. Clones differ in specificity and cytolytic efficacy in chromium-release assays.

(a) Summary of clones giving the patient from whom they were generated and the variants of the dengue GTS epitope recognised by each. Peptide recognition is classified by the percentage of specific lysis of B cells loaded with 0.1 µM of peptide in a standard chromium release assay. +++ 50% or greater lysis, ++ between 20 and 50% lysis, + less than 20% lysis, − no lysis. Cross-reactive clones (b) varied in the extent to which they recognised the epitope variants on pulsed B-cells in CTL lysis assays. Serotype specific clones (c) recognising pD2 were generated from patient MD1413 and those recognising pD3/4 from patient BC307 – all showed less lytic effect at low peptide concentrations than cross-reactive clones.

Figure 4
Figure 4. Most cross-reactive clones show significantly better staining with CD8-null tetramers than serotype-specific clones.

Clones were stained with CD8-null and wild-type A11 tetramers refolded with the DEN3 variant of the GTS epitope. Cross-reactive clones (a) generally showed a smaller drop in fluorescent intensity than DEN3 specific clones which showed much poorer staining with the CD8-null tetramer than with the wild-type (b). The exception was cross-reactive clone 10H5 which showed negligible binding to pD3/4 CD8-null tetramer. No clones showed significant background staining with an unrecognised tetramer (c). pD3/4 tetramer dissociates more rapidly from serotype specific clone D9 than cross-reactive clones E5 or 9F5 (d) in a tetramer dissociation assay.

Figure 5
Figure 5. Cross-reactive clones produce higher levels of both type 1 and type 2 cytokines than serotype-specific clones.

(a) Cytokines produced by cross-reactive (10B3, 9F5, 10H5) and DEN2-specific clones (3H9, 10A4) derived from patient MD1413 stimulated with B cells pulsed with 10 micM pD2 at an E∶T ratio of 5∶1. Cross-reactive clones produced slightly higher levels of each cytokine when stimulated with pD1 or pD3/4 than with pD2. Only pD2 data is shown here to allow comparison of the cross-reactive response with the DEN2 specific clones. (b) Cytokines produced by cross-reactive (E5, C48) and DEN3-specific (D9) clones derived from patient BC307 stimulated with B cells pulsed with 1 micM pD3/4 at an E∶T ratio of 10∶1. pD2 consistently resulted in a lower level of effector function than the same stimulation with pD1 or pD3/4. Black bar: peptide pulsed B cells, white bar: RPMI control pulsed B cells. Data is representative of three experiments. (c, d) IFN-γ and TNF-α production by clones E5 and D9 at an E∶T ratio of 5∶1. Both produce similar quantities when stimulated by cognate peptide at high concentrations. At lower concentrations cross-reactive clone E5 produces up to 4 times more IFN-γ than serotype specific D9. A similar phenomenon is seen with GM-CSF (data not shown). This figure is representative of three independent experiments.

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