The Drosophila lymph gland, the larval hematopoietic organ comprised of prohemocytes and mature hemocytes, has been a valuable model for understanding mechanisms underlying hematopoiesis and immunity. Three types of mature hemocytes have been characterized in the lymph gland: plasmatocytes, lamellocytes, and crystal cells, which are analogous to vertebrate myeloid cells, yet molecular underpinnings of the lymph gland hemocytes have been less investigated. Here, we use single-cell RNA sequencing to comprehensively analyze heterogeneity of developing hemocytes in the lymph gland, and discover previously undescribed hemocyte types including adipohemocytes, stem-like prohemocytes, and intermediate prohemocytes. Additionally, we identify the developmental trajectory of hemocytes during normal development as well as the emergence of the lamellocyte lineage following active cellular immunity caused by wasp infestation. Finally, we establish similarities and differences between embryonically derived- and larval lymph gland hemocytes. Altogether, our study provides detailed insights into the hemocyte development and cellular immune responses at single-cell resolution.

Drosophila hemocytes, like those of mammals, are given rise from two distinctive phases during both the embryonic and larval hematopoiesis. Embryonically derived hemocytes, mostly composed of macrophage-like plasmatocytes, are largely identified by genetic markers. However, the cellular diversity and distinct functions of possible subpopulations within plasmatocytes have not been explored in Drosophila larvae. Here, we show that larval plasmatocytes exhibit differential expressions of Hemolectin (Hml) and Peroxidasin (Pxn) during development. Moreover, removal of plasmatocytes by overexpressing pro-apoptotic genes, hid and reaper in Hml-positive plasmatocytes, feeding high sucrose diet, or wasp infestation results in increased circulating hemocytes that are Hml-negative. Interestingly these Hml-negative plasmatocytes retain Pxn expression, and animals expressing Hml-negative and Pxn-positive subtype largely attenuate growth and abrogate metabolism. Furthermore, elevated levels of a cytokine, unpaired 3, are detected when Hml-positive hemocytes are ablated, which in turn activates JAK/STAT activity in several tissues including the fat body. Finally, we observed that insulin signaling is inhibited in this background, which can be recovered by concurrent loss of upd3. Overall, this study highlights heterogeneity in Drosophila plasmatocytes and a functional plasticity of each subtype, which reaffirms extension of their role beyond immunity into metabolic regulation for cooperatively maintaining internal homeostatic balance.

SUMMARYDrosophila lymph gland, the larval hematopoietic organ comprised of prohemocytes and hemocytes, has been a valuable model for understanding mechanisms underlying hematopoiesis and immunity. Three types of mature hemocytes have been characterized in the lymph gland: plasmatocytes, lamellocytes, and crystal cells, which are analogous to vertebrate myeloid cells. Here, we used single-cell RNA sequencing to comprehensively analyze heterogeneity of developing hemocytes in the lymph gland, and discovered novel hemocyte types, stem-like prohemocytes, and intermediate prohemocytes. Additionally, we identified the emergence of the lamellocyte lineage following active cellular immunity caused by wasp infestation. We unraveled similarities and differences between embryonically derived- and larval lymph gland hemocytes. Finally, the comparison of Drosophila lymph gland hemocytes and human immune cells highlights similarities between prohemocytes and hematopoietic stem cell, and between mature hemocytes and myeloid cells across species. Altogether, our study provides detailed insights on the development and evolution of hematopoiesis at single-cell resolution.

Drosophila hemocytes are akin to mammalian myeloid blood cells that function in stress and innate immune-related responses. A multi-potent progenitor population responds to local signals and to systemic stress by expanding the number of functional blood cells. Here we show mechanisms that demonstrate an integration of environmental carbon dioxide (CO2) and oxygen (O2) inputs that initiate a cascade of signaling events, involving multiple organs, as a stress response when the levels of these two important respiratory gases fall below a threshold. The CO2 and hypoxia-sensing neurons interact at the synaptic level in the brain sending a systemic signal via the fat body to modulate differentiation of a specific class of immune cells. Our findings establish a link between environmental gas sensation and myeloid cell development in Drosophila. A similar relationship exists in humans, but the underlying mechanisms remain to be established.

In order to reduce the time required to identify a match for unrelated donor hematopoietic stem cell transplantation, a one-step DNA sequencing strategy was employed at the time of recruitment. The impact of this strategy on HLA typing resolution and the effect of current registry requirements on resolution and coding of assignments were evaluated. Sanger-based DNA sequencing was used to obtain diploid exons 2 and 3 HLA-A, -B, -C assignments of 2,747 unrelated African American and 1,822 European American volunteers at recruitment. The results demonstrate the high resolution of the approach and challenge several aspects of the current registry typing strategy. Of the 46% of African American and 74% of European American individuals whose HLA typing resulted in alternative genotypes, the majority (≥93%) were predicted to have only a single “common” genotype among the alternatives. The common practice of adding secondary assays to resolve alternative genotype assignments that include more than two antigen groups was also evaluated. While the percentage of assignments with greater than two antigen groups reached as high as 21% (HLA-A in European Americans), only 1.8% of individuals at most carried two common genotypes encompassing three antigen groups. The assignment of NMDP-designated allele codes to the one-pass results reduced the resolution substantially and introduced genotypes that were not included in the laboratory’s assignments. We suggest the alternative strategy of using the exons 2–3 diploid nucleotide sequence as the assignment submitted to the registry with the added benefit of immortalizing the assignment in time regardless of the introduction of novel alleles. To keep pace with current donor selection criteria and with the increasing number of new alleles, it is time to rethink our recruitment typing strategies.