Dengue is a mosquito-borne viral disease transmitted through the bite of the mosquito Aedes aegypti. As per the World Health Organization, an estimated 100-400 million people are infected with the dengue virus (DENV) every year. The disease could be asymptomatic, or in symptomatic cases, the symptoms could range from mild, self-limiting disease to more severe, life-threatening forms such as dengue hemorrhagic fever (DHF) and dengue shock syndrome. The severe forms of the disease could be caused by poorly-neutralising and cross-reactive antibodies.

Additionally, in patients infected with DENV, poor humoral immunity is observed as optimal memory B-cells are not generated. Several studies have highlighted the cytotoxic role of CD4+ T cells in dengue, however, little is known about follicular helper T cells (Tfh cells) that support the antibody-mediated immunity to the DENV.

In a study published in the journal Cell Reports, researchers identified and characterized a distinct subset of peripheral helper CD4+ T cells that contributes to humoral immune responses during dengue infection. The investigation was conducted on 170 adult patients with acute dengue who were hospitalised at the All India Institute of Medical Sciences (AIIMS), New Delhi. Blood samples from the patients were collected in the acute phase and the recovery phase of the disease.

From the blood, peripheral blood mononuclear cells (PBMCs) were isolated, and analysed through flow cytometry. The analyses revealed a unique subset of T follicular helper cells. Typically, T follicular helper cells express a chemokine receptor, CXCR5, on their surface. However, a major fraction of this new subset of cells did not express the CXCR5 receptor. These cells comprised a distinct population, and are called CXCR5⁻ PD-1⁺ CD4⁺ T cells from the patients with severe disease.

These cells also produced the interleukin IL-21, a cytokine that drives B-cell activation and differentiation, among other functions. The cells, through an IL-21 signaling axis, drive B cells to become plasmablasts—short-lived, antibody-producing cells. The massive expansion of the plasmablasts correlates with the severity of the disease.

The researchers further conducted single-cell RNA analysis of the activated CD4+ T cells to understand the diversity of the cell types and the gene expression profiles. The analysis uncovered the presence of ten different clusters representing various functional states, including regulatory, proliferative, and memory cells. Importantly, researchers observed that the CXCR5⁻ PD-1⁺ population consisted of two different functional subtypes—the cytotoxic and helper subsets.

The researchers then performed T cell receptor sequencing analysis to check the clonal relationships within the ten identified clusters. The analyses indicated that the CXCR5⁻ PD-1⁺ CD4⁺ T cells are different from other circulating Tfh cells. They are developmentally and transcriptionally distinct from the Tfh cells and migrate to inflamed tissues.

To demonstrate that the newly identified T cells helped the B cells, the researchers cocultured the T and B cells ex vivo. They observed that both—the peripheral helper CXCR5⁻PD-1⁺ cells and the circulating CXCR5+PD-1⁺ cells drove the B cells to differentiate into plasmablasts. Further, when the activity of IL-21 was blocked, the plasmablast output sharply dropped. Overall, this investigation underscored the collaboration between CXCR5⁻PD-1⁺ cells and the B cells through the IL-21 signaling axis, further indicating that the IL-21 signaling axis could be potentially targeted for desiging treatment strategies.

In dengue, the poorly-neutralising cross-reactive antibodies may lead to a condition called antibody-dependent enhancement (ADE). ADE occurs when a second DENV infection with a different viral serotype causes the previously-developed antibodies to bind to the virus. But since these antibodies cannot neutralise the virus, an antibody-virus complex is formed, which, instead of being killed, enters the immune cells and rapidly replicates, resulting in a more severe form of the disease.

Thus, ADE poses a significant challenge in vaccine development against dengue. The present study is the first report to show the presence of a non-follicular B cell helper subset that accumulates atypically in patients with severe dengue. It has implications for designing effective vaccines and treatments against dengue.