Plasma leakage, a hallmark of severe dengue disease, occurs late after infection and is coincident with viral clearance22, 23
. This, together with evidence that increased disease severity is associated with secondary heterologous DENV infection3–5
, suggests the involvement of cross-reactive DENV-specific memory T and B cells in contributing to an immunopathogenically-mediated clinical outcome.
We found that epitope-specific CD8+
T cell lines isolated from primary DENV immune donors were highly cross-reactive and demonstrated greater recognition of pD1 and pD3/4 variant peptides as compared to pD2. Dong, et al.
also detected significant pD1-3/4 cross-reactivity within this A11 epitope-specific T cell population after secondary DENV infection13
. Though a different DENV-2 epitope variant was used in that study, a majority of their ‘highly cross-reactive’ T cell clones recognized pD1 and pD3/4 with greater potency than the DENV-2 variant.
The predominance of pD1-3/4 cross-reactivity may be explained by the primary structure of these epitope variants. Classic A11-restricted epitopes have a small aliphatic residue at position 2 (P2) and a basic residue at the C terminus24–26
. The epitope used in our study follows this motif since it contains a threonine at P2 as well as a C-terminal arginine. Recently, a secondary anchor residue for A11-restricted epitopes was identified at P6 or P7, which creates a double bulge of neighboring amino acids exposing likely sites of TCR contact27
. Such bulging of the A11 dengue epitope would expose P9, the residue that differs between pD1 or pD3/4 and pD2. However, P9 of a 10-mer epitope from HIV was determined to be the C-terminal MHC anchor residue28
. No matter which side of the MHC-TCR interface interacts with the P9 residue, the non-conservative amino acid change (and corresponding charge difference) between pD1 or pD3/4 (asparagine) and pD2 (aspartate) could drastically affect binding avidity. These epitope variants have been crystallized together with HLA-A*110129
, which indicates tight binding of peptide and MHC. However, three different algorithms predicted that A11 binding for the three peptide variants reflects a hierarchy of pD3/4 > pD1
pD2 (Supplementary Table 1
). We speculate that poor binding of pD2 (compared to pD1 and pD3/4) to either A11 or the TCR may explain its lower functional avidity apparent in our study.
Regardless of their serotype-cross-reactivity, the functional response of all the CD8+
T cell lines we tested revealed a hierarchical induction of MIP-1β > degranulation > TNFα > IFNγ. This is in line with a recent report from our laboratory characterizing PBMC from DENV vaccinees in response to in vitro
stimulation of homologous and heterologous HLA-A*0201-restricted epitope variants12
. Imrie et al.
, however, found that a larger proportion of CD8+
T cell clones specific for a B*5502-restricted epitope on the DENV NS5 protein produced IFNγ compared to TNFα14
. Studies of HIV-specific T cells demonstrated a hierarchical induction of MIP-1β > degranulation > IFNγ > TNFα > IL-230, 31
. Patients who were better able to control their HIV viral loads had higher numbers of T cells exhibiting multiple effector functions in both PBMC30
as well as rectal mucosa31
, suggesting a protective role for polyfunctional CD8+
T cells. While the hierarchical induction of MIP-1β > degranulation > cytokines in virus-specific CD8+
T cells was observed in all of these studies, it is unclear what factors affect the particular order of cytokine production.
Previous studies have shown that varying degrees of TCR-ligand interaction elicit a hierarchical order of response thresholds for different effector functions32–37
. It was determined that, regardless of ligand potency, the extent of TCR engagement as measured by TCR down-regulation dictated the functional response elicited from the T cell. Specifically, cytotoxicity was elicited after very low levels of TCR occupancy by peptide-MHC whereas cytokine responses required higher occupancy for induction. Our data support these findings in that cytotoxicity (measured by target cell lysis and degranulation) was induced at the lowest concentrations of full agonist peptides used as well as high concentrations of partial agonist peptides. TNFα and IFNγ production was only seen after stimulation with full agonist peptides, and the proportion of cells producing these cytokines increased with increasing concentrations of those peptides, supporting the idea that greater TCR occupancy triggers cytokine production. Additionally, phosphoflow assays performed in our study confirm that low TCR occupancy qualitatively alters peptide-induced signaling pathways, which helps explain differential effector responses. This is the first time phosphoflow has been utilized to demonstrate variations in peptide-induced signaling in T cells and has proven to be an effective tool for measuring such differences. Altogether our data argues that production of the chemokine MIP-1β has an even lower signal threshold than cytotoxicity, supporting other studies which suggest MIP-1β is a more sensitive measure of antigen-specific cells than IFNγ30,38
In addition to lifelong immunity to the currently-infecting serotype, primary DENV infection induces transient but protective immunity to heterologous DENV serotypes in patients for at least two months after illness39
. Our patient samples were collected eight months or less after primary infection. It is possible that the cross-reactive T cell responses we observed might not represent stable, long term T cell memory. Analysis of memory T cell responses after longer intervals would be complicated by the substantial potential for re-exposure to DENV in individuals living in DENV-endemic areas. However, we have detected cross-reactive T cell responses in vaccine recipients several years after immunization12, 40, 41
suggesting that primary exposure to the virus generates a long-lived cross-reactive memory T cell response.
Our findings support epidemiological evidence which indicates that the order of DENV serotypes with which a particular individual is infected plays a critical role in the outcome of the secondary immune response. The data suggest that, for the response to this A11-restricted epitope, DENV-1 infection followed by DENV-3 infection, or vice versa, would trigger the activation of cross-reactive T cells that undergo a vigorous polyfunctional response involving IFNγ production. On the other hand, a secondary DENV-2 infection may elicit cross-reactive, poorly responding T cells that are skewed toward MIP-1β production. Increased MIP-1β production at the site of infection may augment IFNγ and TNFα production by T cells42
activated by other viral epitopes and increase TNFα production by monocytes43
elevating the circulating levels of these cytokines and ultimately contributing to immunopathology. Secondary DENV-2 infection has been observed as a risk factor for increased disease severity in epidemiological studies in Asia3, 4
Our study characterized effector functions in CD8+
T cells specific for a single epitope, whereas the overall T cell response would reflect responses to multiple epitopes with varying patterns of serotype-cross-reactivity. The clinical outcome in a given DENV-infected individual would also be expected to depend on other host characteristics including age, prior infection history, HLA alleles, and other genetic variables11
. Although our study was limited to three patients, the subjects varied with regard to the infecting serotype, age, illness grade as well as genetic background. We isolated many cell lines from each patient that showed similar patterns of serotype-cross-reactivity to this epitope. Further studies on individuals infected with other DENV serotypes, experiencing different illnesses and with different genetic backgrounds than our donors will add to our understanding of the contribution of this cross-reactive epitope-specific T cell population to disease.
This study is the first to demonstrate substantial cross-reactivity of antigen-specific T cells generated in response to naturally-acquired primary DENV infection. We provide evidence that the DENV-immune T cell repertoire exhibits altered functional capabilities that are dependent on the particular serotype of infection. Investigations into the role of heterologous epitope variants in triggering memory T cell responses in donors with primary DENV infection help to define parts of the preexisting immune response that may predispose a person towards developing severe disease. An effective vaccine or treatment regimen will have to consider the effects of prior flavivirus immunity on disease outcome and will hopefully lead to the eradication of this widespread viral threat.