Fig 2: The proliferation rate and cytokines production of antigen experienced CD4+ and CD8+ T cells co-cultured with anti-CD300a antibodies treated and parasites infected BMDCs. To quantify T cells proliferation, the CFSE stained pan T cells isolated from splenocytes of animals after 14 days post-infection, were cultured with BMDCs (1:10; DCs to T cells ratio). A representation of gating strategies to quantify the percent of antigen experienced CFSE+ CD4+/CD8+ T cells is depicted in panels (A-C). The CD4+ and CD8+ T cells cultured with anti-CD300a treated and parasite infected BMDCs were found more proliferative in comparison to the cells cultured with untreated BMDCs (D). The CD300a receptor blocking significantly increased the production of IFN-? and IL-12 in antigen experienced T cells (E, F). The blocking of CD300as receptors also enhanced IFN-?+/IL-12+ producing T cells (G), and improved their IFN-?/IL-10 ratio (H). The cytokines positive T cells were measured on CD4+CD44+ T cells that were gated on CD3+ T cells and isotype antibodies were used as a control to determine statistical significance.

Fig 3: The CD300a mRNA and protein expression levels in the spleen of L. donovani infected mice. Mice (n=6 per group), infected with 3×106 stationary phase promastigotes via tail vein, were evaluated post 7 (D-7), 14 (D-14), and 21 (D-21) days of infection. The expression levels of CD300a mRNA (A) and protein (B) were significantly elevated in infected mice with the passage of time as compared to day 7. (C). The number of CD300a expressing CD11c+ dendritic cells in the spleen, measured as Mean Fluorescent Intensity (MFI) by flow cytometry, were found significantly increased at all time points post-infection. An unvarying expression of CD300a was observed in the spleen tissues (E) that increased with the passage of time in the infected animals (D). The uninfected (UI) animals were used as a control for comparison to determine statistical significance (p-value).

Fig 4: The memory phenotypes, i.e., CCR7+CD62L+CD4+ T cells in lymph nodes after CD300a blocking in infected animals. The number of CD4+ T cells expressing effector and central memory surface markers i.e., CCR7+ (A), CD62L+ (B) along with double positives CCR7+CD62L+ central memory CD4+ T cells (C) were significantly higher at D14 and D21 in antibodies treated animals as compared to those who received isotypic antibodies.

Fig 5: A schematic model for the role of CD300a receptor in parasitic infectivity, APCs and T cells effector functions, and memory generation during Leishmania infection. Based on observations made in the current study, it may be surmised that the parasite induces CD300a receptors on the host’s phagocytic and antigen presenting cells to counter the defense machinery, which eventually facilitates the survival of parasites. The blocking of CD300a receptors increases APCs function and promotes antigen specific T cells activation and differentiation required to establish early protective immunity. Thus, strategies involving CD300a receptor manipulation may prove advantageous to curb the survival of Leishmania in their mammalian host and also can boost antigen-specific immune responses.