Phenotyping of peripheral blood mononuclear cells during acute dengue illness demonstrates infection and increased activation of monocytes in severe cases compared to classic dengue fever

Phenotyping of peripheral blood mononuclear cells during acute dengue illness demonstrates infection and increased activation of monocytes in severe cases compared to classic dengue fever. CD8+ T lymphocytes, but preactivation of T cells reduces the susceptibility of T HLI 373 cells to DV infection. Interestingly, the cytotoxicity-inducing protein granzyme A is highly secreted by human CD4+ but not CD8+ T cells after exposure to DV and induces the secretion of inflammatory mediators, apoptosis, and polyclonal B cell activation, which contribute to vascular leakage and DV-induced disease (5,C8). In addition to the above-mentioned cells, T lymphocytes are a major population activated during dengue fever (9, 10). Previous reports have indicated that CD4+ and CD8+ T cells play a role in the control of DV infection, mostly due to T cell-dependent cytotoxicity against virus-infected cells (11, 12). In support of this concept, CD8+ T cell activation and proliferation are inversely correlated with dengue viremia and appear to occur late in the course of DV infection (13). In contrast, low-affinity anti-DV T cells, induced during secondary heterotypic infection, contribute to the high viral load CBLL1 and intense inflammatory cytokine secretion observed in severe dengue cases (14). Moreover, activated CD4+ and CD8+ T cells have previously been found to be associated with hemorrhagic disease (10, 13, 15). This suggests that while T cells may contribute to controlling DV replication, they could also be involved in the pathogenesis of the disease (3, 16). It is possible that DV directly infects human T cells and affects their functions. It has been shown that human T leukemia cells and T cell lines can be infected by DV serotype 2 (17, 18) and in humanized mice (19), suggesting a possible interaction between this flavivirus and human T cells. However, using flow cytometry, two reports have demonstrated that human T cells are not infected by DV (20) or (21). Nevertheless, whether DV directly interacts with primary human T cells and the possible consequences of these interactions during DV infection remain largely unknown. To gain insight into possible DV-T cell interactions, we used a series of HLI 373 virology- and immunology-based assays with primary human CD4+ and CD8+ T cells exposed to DV serotypes 1 to 4. We observed that naive primary human T lymphocytes (CD4+ and CD8+) are permissive for DV infection and support viral replication, as well as the synthesis of infectious virus particles. Additionally, after infection by DV, T lymphocytes became activated and CD4+, but not CD8+, T cells secreted granzyme A (GzmA). Despite being infected by DV, T lymphocytes were resistant to DV-induced apoptosis. Additionally, using peripheral blood mononuclear cells (PBMCs) from acutely infected dengue patients, we confirmed the susceptibility of CD4+ and CD8+ T cells to DV. Together, our observations reveal a novel DV-host interaction that could contribute to the understanding of dengue pathogenesis. RESULTS DV infects and replicates in CD4+ and CD8+ T lymphocytes through interaction with the heparan sulfate moiety. Because dengue fever patients have previously been shown to display enhanced T cell activation (10, 13, 15), we first asked whether DV directly interacts with T lymphocytes. For this assessment, we infected PBMCs with different multiplicities of infection (MOIs) of DV3 and observed that CD4+ and CD8+ T cells are susceptible to dengue virus infection (Fig. 1). In addition, kinetic experiments using PBMCs from healthy donors confirmed the infection of CD4+ and CD8+ T lymphocytes by DV3 (see Fig. S1 in the supplemental material). Thus, PBMCs from healthy donors were exposed to the four DV serotypes (MOI of 10) and, after 5 days postinfection, virus infection was measured by means of intracellular staining of the virus envelope protein through flow cytometry (22). As previously shown (7, 23, 24), HLI 373 B lymphocytes (CD19+) and monocytes (CD14+) were infected by DV serotypes 1 to 4 (Fig. S2A, D, and E). Similarly, CD4+ and CD8+ T cells were found to be infected by the four DV serotypes (Fig. S2A to C). Furthermore, when purified CD4+ and CD8+ T cell populations from 6 HLI 373 healthy donors (Fig. S2G and H) were infected with DV, similar results were observed (Fig. 2A to ?toE).E). It is noteworthy that confocal microscopy of purified T cell populations exposed to DV confirmed that CD4+ and CD8+ T lymphocytes were infected by DV (Fig. 2D and ?andE).E). All DV serotypes presented similar levels of infectivity in T cells (Fig. 2B and ?andCC). Open in a separate window FIG 1 Infection of CD4+ and CD8+ T lymphocytes with different MOIs of DV3. (A) Representative flow cytometry density plot data showing the results for mock infection and infection of CD4+ and.