Our results demonstrate similar frequencies of serotype-cross-reactive T cells ex vivo in naturally-infected patients with primary and secondary DENV infections. T cell functional responses to heterologous DENV epitopes were observed previously in some subjects following primary DENV infections8,28
, but this is the first demonstration of serotype-cross-reactivity in primary infection based on binding of heterotypic tetramers ex vivo, and illustrates the breadth of the phenomenon. Mongkolsapaya et al. found that A11-NS3133
-specific T cells in patients with secondary DENV infections preferentially stained ex vivo with pMHC tetramers corresponding to DENV serotypes heterologous to the infecting serotype13
. We used a similar approach, but extended our analysis to primary DENV infections, an important comparison group absent in the earlier study. Preferential binding to the pD1 tetramer (followed by the pD3/4 tetramer and, finally, the pD2 tetramer) was also seen in our previous study of T cell lines, which we postulated could be explained by their predicted MHC binding (pD3/4 ≥ pD1
. Heterotypic tetramer binding for this epitope thus does not reflect the history of prior infection with other DENV serotypes; other possible explanations for this phenomenon are heterologous immunity from prior unrelated infections29
, characteristics of the T cell repertoire30
, or inherent immunogenicity of different epitope variants8,31
. It is possible that preferential binding to the pD1 tetramer was a reflection of it being the most stable of the three tetramers. However, this preferential binding would apply equally to the entire dataset, which would not affect our ability to detect differences in the overall pattern of tetramer staining between various subject groups. Despite donor-to-donor variability, we did note some similarities in patterns of tetramer staining among individuals with the same infection status (DENV serotype, primary versus secondary). This suggests that the sequence of infection does play a role in shaping the DENV-specific memory T cell repertoire, although other factors are also important.
Mongkolsapaya et al. reported low but detectable frequencies of A11-NS3133
-specific T cells during acute secondary DENV infection in Thai children which peaked in early convalescence13
. In contrast, Dung et al. reported that A11-NS3133
-specific T cells were undetectable until after the development of plasma leakage among infected Vietnamese children25
. Our data (for both the A11-NS3133
epitopes) are clearly more consistent with the earlier report, and, in fact, several of our subjects had very high frequencies of (activated) epitope-specific T cells during acute infection. These immune responses occurring prior to the onset of plasma leakage therefore have the potential (i.e. are available) to contribute to disease pathogenesis. The finding of antigen-specific T cell expansion and activation during acute illness is also more consistent with observations in other viral diseases27,32,33
and with other evidence of T cell activation during acute DENV infection20,23,26
. The divergent findings of Dung et al.
may reflect differences in experimental technique; a more intriguing possibility is that the expansion of A11-NS3133
-specific T cells might differ between Thai and Vietnamese patients. In this regard, it is of interest that an association with dengue disease severity has been observed for HLA-A11 in our Thai cohort34
but was not noted in a study in Vietnam35
despite the similar genetic backgrounds of these two populations.
We found that the frequency of epitope-specific T cells peaked earlier in subjects with primary infection than in those with secondary infection. Prior studies in which DENV-specific T cells were tracked over time had not differentiated between subjects with primary and secondary infections. Although this result was not anticipated, it is consistent with findings in DENV infection of BALB/c mice31
. More rapid activation of memory T cells would not necessarily correspond to an earlier peak T cell frequency, since sustained proliferation signals balanced by apoptosis13,36
would affect the timing of peak responses to a greater extent. Future studies need to incorporate analysis of these mechanisms as well as other immunomodulatory signals such as regulatory T cells37
The magnitude of A11-NS3133
-specific T cells did not correlate with disease severity in this cohort. This held true whether the data were analyzed according to clinical diagnosis (DF versus DHF) or continuous measures of disease severity such as pleural effusion index, hemoconcentration, or platelet nadir. A similar lack of association was reported in a study in Vietnam25
. Other studies had reported higher frequencies of DENV-specific T cells in patients with DHF, but these associations were found at 2 weeks13,14
or 6 months24
The interpretation of our data is subject to several limitations. Although our patient cohort was relatively large, the small number of HLA-A11+
subjects with the same DENV serotype, serologic response (primary/secondary infection), and clinical outcome (DF/DHF) limits the statistical power for important subgroup analyses, and data from HLA association studies suggest that the influence of T cell responses may not be the same for all four serotypes34
. Additionally, although subjects were followed daily during acute illness, adequate specimens were not available for all of these flow cytometry studies from all time points for all subjects. Given that rapid changes were observed in tetramer-positive T cell frequencies during acute infection, it is likely that peak frequencies were missed in some subjects. Furthermore, few of the DHF patients in our study experienced shock (DHF grades 3 or 4). As Mongkolsapaya et al. found the highest frequencies of tetramer-positive T cells in patients with shock13,14
, it is possible that the milder disease in our study cohort concealed a relationship to the most severe disease. Alternatively, we cannot exclude the possibility that the A11-NS3133
epitope is not representative of the global DENV-specific CD8+
T cell response. Duangchinda et al. recently showed higher cytokine responses in children with DHF compared to DF when overlapping peptides covering the entire NS3 protein were used38
. However, that study only analyzed PBMC collected several weeks post-infection and the same association was not detected by Simmons et al.39
. Finally, human studies are limited to analysis of blood samples and we may have missed pathogenic T cells that were bound to the endothelium or located in other tissues.
In summary, our data points to a complex picture of T cell involvement in DENV infection. We found a selective activation and expansion of DENV-specific T cells during acute infection and a diverse pattern of serotype-cross-reactivity in both primary and secondary infections. These findings will need to be taken into account in future studies of T cell responses to natural DENV infection and/or vaccines.