Cytotoxic Vδ2+ T Cell Subsets Expand in Response to Malaria in Human Tonsil and Spleen Organoids

Vaccine effectiveness against malaria is dramatically reduced in malaria-exposed compared to malaria-naïve populations, potentially due to altered immune responses in secondary lymphoid organs following repeated infection. Newly developed human tonsil and spleen organoids, which replicate key features of B and T cell immunity, provide an exciting opportunity to overcome challenges of other models and to improve our understanding of innate-adaptive interactions in lymphoid tissue. The objectives of this study were to use these organoids to investigate the impact of malaria parasites on 1) cells within lymphoid tissues and 2) responses to a heterologous antigen. When we exposed organoids from malaria-naïve donors to Plasmodium falciparum-infected red blood cells (iRBC), we observed that iRBC exposure did not disrupt organoid formation and significantly increased Vδ2 + γδ T cell frequencies in both tonsil and spleen organoids at multiple timepoints. Single-cell RNA/TCR sequencing revealed that iRBC-responsive Vδ2 + T cells in organoids were clonally expanded and exhibited activated, cytotoxic phenotypes with upregulated expression of granzymes, interferon-stimulated genes, and antigen presentation machinery. TCR repertoire analysis demonstrated that malaria exposure drove clonal expansion of cytotoxic Vδ2 + T cells, contrasting with the diverse, smaller clones observed in control conditions. To validate these findings, we analyzed tonsils from Ugandan children with asymptomatic parasitemia and found expanded Vδ2 + T cells with enhanced cytotoxic potential compared to uninfected controls. When we tested whether malaria pre-exposure affected subsequent recall responses to influenza vaccine, malaria pre-exposure or γδ T cell depletion did not significantly alter cellular frequencies or influenza-specific antibody responses in most donors, though modest reductions were observed in some individuals. This work demonstrates the utility of human lymphoid organoids for studying malaria-host interactions and provides novel insights into Vδ2 + T cell biology, including evidence for clonal expansion and cytotoxic differentiation in response to malaria parasites within secondary lymphoid tissues.