The Atlantic cod’s unusual immune system, entirely lacking the Major Histocompatibility class II pathway, has prompted intriguing questions about what mechanisms are used to combat bacterial infections and how immunological memory is generated. By single-cell RNA sequencing we here report an in-depth characterisation of cell types found in immune tissues, the spleen and peripheral blood leukocytes of Atlantic cod. Unbiased transcriptional clustering revealed eleven distinct immune cell signatures. Resolution at the single cell level enabled characterisation of the major cell subsets including the cytotoxic T cells, B cells, erythrocytes, thrombocytes, neutrophils, and macrophages. Additionally, to our knowledge we are the first to uncover cell subsets in Atlantic cod which may represent dendritic cells, natural killer-like cells, and a population of cytotoxic cells expressing GATA-3 , a master transcription factor of T helper 2 cells. We further identify putative gene markers for each cluster and describe the relative proportions of each cell type in the spleen and peripheral blood leukocytes. Of the major haematopoietic cell populations, the lymphocytes make up 55 and 68% of the spleen and peripheral blood leukocytes respectively, while the myeloid cells make up 45 and 32%. By single-cell analysis, this study provides the most detailed molecular and cellular characterisation of the immune system of the Atlantic cod so far.

Teleost adaptive immune systems have evolved with more flexibility than previously assumed. A particularly enigmatic system to address immune system modifications in the evolutionary past is represented by the Syngnathids, the family of pipefishes, seahorses and seadragons. These small fishes with their unique male pregnancy have lost the spleen as an important immune organ as well as a functional major histocompatibility class II (MHC II) pathway. How these evolutionary changes have impacted immune cell population dynamics have up to this point remained unexplored. Here, we present the first immune cell repertoire characterization of a syngnathid fish (Syngnathus typhle) using single-cell transcriptomics. Gene expression profiles of individual cells extracted from blood and head-kidney clustered in twelve putative cell populations with eight belonging to those with immune function. Upregulated cell marker genes identified in humans and teleosts were used to define cell clusters. While the suggested loss of CD4+ T-cells accompanied the loss of the MHC II pathway was supported, the upregulation of specific subtype markers within the T-cell cluster indicates subpopulations of regulatory T-cells (il2rb) and cytotoxic T-cells (gzma). Utilizing single-cell RNA sequencing this report is the first to characterize immune cell populations in syngnathids and provides a valuable foundation for future cellular classification and experimental work within the lineage.

Atlantic Cod (Gadus morhua) has lost the major histocompatibility complex class II presentation pathway. We recently identified CD8-positive T cells, B cells, and plasma cells in cod, but further characterisation of lymphocyte subsets is needed to elucidate immune adaptations triggered by the absence of CD4-positive T lymphocytes. Here, we use single-cell RNA sequencing to examine the lymphocyte heterogeneity in Atlantic cod spleen. We describe five T cell subsets and eight B cell subsets and propose a B cell trajectory of differentiation. Notably, we identify a subpopulation of T cells that are CD8-negative. Most of the CD8-negative T lymphocytes highly express the homologue of monocyte chemotactic protein 1b, and another subset of CD8-negative T lymphocytes express the homologue of the scavenger receptor m130. Uncovering the multiple lymphocyte cell sub-clusters reveals the different immune states present within the B and T cell populations, building a foundation for further work.

Innate lymphoid cells (ILCs) are important for tissue immune homeostasis, and are thoroughly characterized in mice and humans. Here, we have performed in-depth characterization of rat ILCs. Rat ILCs were identified based on differential expression of transcription factors and lack of lineage markers. ILC3s represented the major ILC population of the small intestine, while ILC2s were infrequent but most prominent in liver and adipose tissue. Two major subsets of group 1 ILCs were defined. Lineage -T-bet + Eomes + cells were identified as conventional NK cells, while lineage -T-bet + Eomescells were identified as the probable rat counterpart of ILC1s based on their selective expression of the ILC marker CD200R. Rat ILC1s were particularly abundant in liver and intestinal tissues, and were functionally similar to NK cells. Single-cell transcriptomics of spleen and liver cells confirmed the main division of NK cells and ILC1-like cells, and demonstrated Granzyme A as an additional ILC1 marker. We further report differential distributions of NK cells and ILCs along the small and large intestines, and the association of certain bacterial taxa to frequencies of ILCs. In conclusion, we provide a framework for future studies of ILCs in diverse rat experimental models, and novel data on the potential interplay between commensals and intestinal ILCs.

Atlantic Cod (Gadus morhua) has lost the major histocompatibility complex class II presentation pathway. We recently identified CD8-positive T cells, B cells, and plasma cells in cod, but further characterization of lymphocyte subsets are needed to elucidate immune adaptations triggered by the absence of CD4-positive T lymphocytes. Here we use single-cell RNA sequencing to examine the lymphocyte heterogeneity in Atlantic cod spleen. We describe five TCR-positive T cell subsets and eight BCR-positive B cell subsets and propose a B cell trajectory of differentiation. Notably, we identify a significant subpopulation of T cells that are CD8-negative. Most of the CD8-negative T lymphocytes highly express the homolog of monocyte chemotactic protein 1b, and another subset of CD8-negative T lymphocytes express the homolog of the scavenger receptor m130. Uncovering the multiple lymphocyte cell sub-clusters reveals the different immune states present within the B and T cell populations, building a foundation for further work.